High molecular weight surface proteins of non-typeable haemophilus
High molecular weight surface proteins of non-typeable Haemophilus influenzae which exhibit immunogenic properties and genes encoding the same are described. Specifically, genes coding for two immunodominant high molecular weight proteins, HMW1 and HMW2, have been cloned, expressed and sequenced, while genes coding for high molecular proteins HMW3 and HMW4 have been cloned, expressed and partially sequenced.
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This invention relates to high molecular weight proteins of non-typeable haemophilus.
BACKGROUND TO THE INVENTIONNon-typeable Haemophilus influenzae are non-encapsulated organisms that are defined by their lack of reactivity with antisera against known H. influenzae capsular antigens.
These organisms commonly inhabit the upper respiratory tract of humans and are frequently responsible for infections, such as otitis media, sinusitis, conjunctivitis, bronchitis and pneumonia. Since these organisms do not have a polysaccharide capsule, they are not controlled by the present Haemophilus influenzae type b (Hib) vaccines, which are directed towards Hib bacterial capsular polysaccharides. The non-typeable strains, however, do produce surface antigens that can elicit bactericidal antibodies. Two of the major outer membrane proteins, P2 and P6, have been identified as targets of human serum bactericidal activity. However, it has been shown that the P2 protein sequence is variable, in particular in the non-typeable Haemophilus strains. Thus, a P2-based vaccine would not protect against all strains of the organism.
There have previously been identified by Barenkamp et al (Pediatr. Infect. Dis. J., 9:333-339, 1990) a group of high-molecular-weight (HMW) proteins that appeared to be major targets of antibodies present in human convalescent sera. Examination of a series of middle ear isolates revealed the presence of one or two such proteins in most strains. However, prior to the present invention, the structures of these proteins were unknown as were pure isolates of such proteins.
SUMMARY OF INVENTIONThe inventors, in an effort to further characterize the high molecular weight (HMW) Haemophilus proteins, have cloned, expressed and sequenced the genes coding for two immunodominant HMW proteins (designated HMW1 and HMW2) from a prototype non-typeable Haemophilus strain and have cloned, expressed and almost completely sequenced the genes coding for two additional immunodominant HMW proteins (designated HMW3 and HMW4) from another non-typeable Haemophilus strain.
In accordance with one aspect of the present invention, therefore, there is provided an isolated and purified gene coding for a high molecular weight protein of a non-typeable Haemophilus strain, particularly a gene coding for protein HMW1, HMW2, HMW3 or HMW4, as well as any variant or fragment of such protein which retains the immunological ability to protect against disease caused by a non-typeable Haemophilus strain. In another aspect, the invention provides a high molecular weight protein of non-typeable Haemophilus influenzae which is encoded by these genes.
BRIEF DESCRIPTION OF DRAWINGSFIG. 1 is a DNA sequence of a gene coding for protein HMW1 (SEQ ID NO: 1);
FIG. 2 is a derived amino acid sequence of protein HMW1 (SEQ ID NO: 2);
FIG. 3 is a DNA sequence of a gene coding for protein HMW2 (SEQ ID NO: 3);
FIG. 4 is a derived amino acid sequence of HMW2 (SEQ ID NO: 4);
FIG. 5A shows restriction maps of representative recombinant phages which contained the HMW1 or HMW2 structural genes, the locations of the structural genes being indicated by the shaded bars;
FIG. 5B shows the restriction map of the T7 expression vector pT7-7;
FIG. 6 contains the DNA sequence of a gene cluster for the hmw1 gene (SEQ ID NO: 5), comprising nucleotides 351 to 4958 (ORF a) (as in FIG. 1), as well as two additional downstream genes in the 3' flanking region, comprising ORFs b, nucleotides 5114-6748, and c nucleotides 7062-9011;
FIG. 7 contains the DNA sequence of a gene cluster for the hmw2 gene (SEQ ID NO: 6), comprising nucleotides 792 to 5222 (ORF a) (as in FIG. 3), as well as two additional downstream genes in the 3' flanking region, comprising ORFs b, nucleotides 5375-7009, and c, nucleotides 7249-9198;
FIG. 8 is a partial DNA sequence of a gene coding for protein HMW3 (SEQ ID NO: 7);
FIG. 9 is a partial DNA sequence of a gene coding for protein HMW4 (SEQ ID NO: 8); and
FIG. 10 is a comparison table for the derived amino acid sequence for proteins HMW1, HMW2, HMW3 and HMW4.
GENERAL DESCRIPTION OF INVENTIONThe DNA sequences of the genes coding for HMW1 and HMW2, shown in FIGS. 1 and 3 respectively, were shown to be about 80% identical, with the first 1259 base pairs of the genes being identical. The derived amino acid sequences of the two HMW proteins, shown in FIGS. 2 and 4 respectively, are about 70% identical. Furthermore, the encoded proteins are antigenically related to the filamentous hemagglutinin surface protein of Bordetella pertussis. A monoclonal antibody prepared against filamentous hemagglutinin (FHA) of Bordetella pertussis was found to recognize both of the high molecular weight proteins. This data suggests that the HMW and FHA proteins may serve similar biological functions. The derived amino acid sequences of the HMW1 and HMW2 proteins show sequence similarity to that for the FHA protein. It has further been shown that these antigenically-related proteins are produced by the majority of the non-typeable strains of Haemophilus. Antisera raised against the protein expressed by the HMW1 gene recognizes both the HMW2 protein and the B. pertussis FHA. The present invention includes an isolated and purified high molecular weight protein of non-typeable haemophilus which is antigenically related to the B. pertussis FHA, which may be obtained from natural sources or produced recombinantly.
A phage genomic library of a known strain of non-typeable Haemophilus was prepared by standard methods and the library was screened for clones expressing high molecular weight proteins, using a high titre antiserum against HMW's. A number of strongly reactive DNA clones were plaque-purified and sub-cloned into a T7 expression plasmid. It was found that they all expressed either one or the other of the two high-molecular-weight proteins designated HMW1 and HMW2, with apparent molecular weights of 125 and 120 kDa, respectively, encoded by open reading frames of 4.6 kb and 4.4 kb, respectively.
Representative clones expressing either HMW1 and HMW2 were further characterized and the genes isolated, purified and sequenced. The DNA sequence of HMW1 is shown in FIG. 1 and the corresponding derived amino acid sequence in FIG. 2. Similarly, the DNA sequence of HMW2 is shown in FIG. 3 and the corresponding derived amino acid sequence in FIG. 4. Partial purification of the isolated proteins and N-terminal sequence analysis indicated that the expressed proteins are truncated since their sequence starts at residue number 442 of both full length HMW1 and HMW2 gene products.
Subcloning studies with respect to the hmw1 and hmw2 genes indicated that correct processing of the HMW proteins required the products of additional downstream genes. It has been found that both the hmw1 and hmw2 genes are flanked by two additional downstream open reading frames (ORFs), designated b and c, respectively, (see FIGS. 6 and 7).
The b ORFs are 1635 bp in length, extending from nucleotides 5114 to 6748 in the case of hmw1 and nucleotides 5375 to 7009 in the case of hmw2, with their derived amino acid sequences 99% identical. The derived amino acid sequences demonstrate similarity with the derived amino acid sequences of two genes which encode proteins required for secretion and activation of hemolysins of P. mirabilis and S. marcescens.
The c ORFs are 1950 bp in length, extending from nucleotides 7062 to 9011 in the case of hmw1 and nucleotides 7249 to 9198 in the case of hmw2, with their derived amino acid sequences 96% identical. The hmw1 c ORF is preceded by a series of 9 bp direct tandem repeats. In plasmid subclones, interruption of the hmw1 b or c ORF results in defective processing and secretion of the hmw1 structural gene product.
The two high molecular weight proteins have been isolated and purified and shown to be partially protective against otitis media in chinchillas and to function as adhesins. These results indicate the potential for use of such high molecular proteins and structurally-related proteins of other non-typeable strains of Haemophilus influenzae as components in non-typeable Haemophilus influenzae vaccines.
Since the proteins provided herein are good cross-reactive antigens and are present in the majority of non-typeable Haemophilus strains, it is evident that these HMW proteins may become integral constituents of a universal Haemophilus vaccine. Indeed, these proteins may be used not only as protective antigens against otitis, sinusitis and bronchitis caused by the non-typeable Haemophilus strains, but also may be used as carriers for the protective Hib polysaccharides in a conjugate vaccine against meningitis. The proteins also may be used as carriers for other antigens, haptens and polysaccharides from other organisms, so as to induce immunity to such antigens, haptens and polysaccharides.
The nucleotide sequences encoding two high molecular weight proteins of a different non-typeable Haemophilus strain (designated HMW3 and HMW4) have been largely elucidated, and are presented in FIGS. 8 and 9. HMW3 has an apparent molecular weight of 125 kDa while HMW4 has an apparent molecular weight of 123 kDa. These high molecular weight proteins are antigenically related to the HMW1 and HMW2 proteins and to FHA. Sequence analysis of HMW3 is approximately 85% complete and of HMW4 95% complete, with short stretches at the 5'-ends of each gene remaining to be sequenced.
FIG. 10 contains a multiple sequence comparison of the derived amino acid sequences for the four high molecular weight proteins identified herein. As may be seen from this comparison, stretches of identical peptide sequence may be found throughout the length of the comparison, with HMW3 more closely resembling HMW1 and HMW4 more closely resembling HMW2. This information is highly suggestive of a considerable sequence homology between high molecular weight proteins from various non-typeable Haemophilus strains.
In addition, mutants of non-typeable H. influenzae strains that are deficient in expression of HMW1 or HMW2 or both have been constructed and examined for their capacity to adhere to cultured human epithelial cells. The hmw1 and hmw2 gene clusters have been expressed in E. coli and have been examined for in vitro adherence. The results of such experimentation demonstrate that both HMW1 and HMW2 mediate attachment and hence are adhesins and that this function is present even in the absence of other H. influenzae surface structures.
With the isolation and purification of the high molecular weight proteins, the inventors are able to determine the major protective epitopes by conventional epitope mapping and synthesize peptides corresponding to these determinants to be incorporated in fully synthetic or recombinant vaccines. Accordingly, the invention also comprises a synthetic peptide having an amino acid sequence corresponding to at least one protective epitope of a high molecular weight protein of a non-typeable Haemophilus influenzae. Such peptides are of varying length that constitute portions of the high-molecular-weight proteins, that can be used to induce immunity, either directly or as part of a conjugate, against the relative organisms and thus constitute vaccines for protection against the corresponding diseases.
The present invention also provides any variant or fragment of the proteins that retains the potential immunological ability to protect against disease caused by non-typeable Haemophilus strains. The variants may be constructed by partial deletions or mutations of the genes and expression of the resulting modified genes to give the protein variations.
EXAMPLES Example 1Non-typeable H.influenzae strains 5 and 12 were isolated in pure culture from the middle ear fluid of children with acute otitis media. Chromosomal DNA from strain 12, providing genes encoding proteins HMW1 and HMW2, was prepared by preparing Sau3A partial restriction digests of chromosomal DNA and fractionating on sucrose gradients. Fractions containing DNA fragments in the 9 to 20 kbp range were pooled and a library was prepared by ligation into .lambda.EMBL3 arms. Ligation mixtures were packaged in vitro and plate-amplified in a P2 lysogen of E. coli LE392.
For plasmid subcloning studies, DNA from a representative recombinant phage was subcloned into the T7 expression plasmid pT7-7, containing the T7 RNA polymerase promoter .PHI.10, a ribosome-binding site and the translational start site for the T7 gene 10 protein upstream from a multiple cloning site (see FIG. 5B).
DNA sequence analysis was performed by the dideoxy method and both strands of the HMW1 gene and a single strand of the HMW2 gene were sequenced.
Western immunoblot analysis was performed to identify the recombinant proteins being produced by reactive phage clones. Phage lysates grown in LE392 cells or plaques picked directly from a lawn of LE392 cells on YT plates were solubilized in gel electrophoresis sample buffer prior to electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed on 7.5% or 11% polyacrylamide modified Laemmli gels. After transfer of the proteins to nitrocellulose sheets, the sheets were probed sequentially with an E. coli-absorbed human serum sample containing high-titer antibody to the high-molecular-weight proteins and then with alkaline phosphatase-conjugated goat anti-human immunoglobulin G (IgG) second antibody. Sera from healthy adults contains high-titer antibody directed against surface-exposed high-molecular-weight proteins of non-typeable H. influenzae. One such serum sample was used as the screening antiserum after having been extensively absorbed with LE392 cells.
To identify recombinant proteins being produced by E. coli transformed with recombinant plasmids, the plasmids of interest were used to transform E. coli BL21 (DE3)/pLysS. The transformed strains were grown to an A.sub.600 of 0.5 in L broth containing 50 .mu.g of ampicillin per ml. IPTG was then added to 1 mM. One hour later, cells were harvested, and a sonicate of the cells was prepared. The protein concentrations of the samples were determined by the bicinchoninic acid method. Cell sonicates containing 100 .mu.g of total protein were solubilized in electrophoresis sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The nitrocellulose was then probed sequentially with the E. coli-absorbed adult serum sample and then with alkaline phosphatase-conjugated goat anti-human IgG second antibody.
Western immunoblot analysis also was performed to determine whether homologous and heterologous non-typeable H. influenzae strains expressed high-molecular-weight proteins antigenically related to the protein encoded by the cloned HMW1 gene (rHMW1). Cell sonicates of bacterial cells were solubilized in electrophoresis sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Nitrocellulose was probed sequentially with polyclonal rabbit rHMW1 antiserum and then with alkaline phosphatase-conjugated goat anti-rabbit IgG second antibody.
Finally, Western immunoblot analysis was performed to determine whether non-typeable Haemophilus strains expressed proteins antigenically related to the filamentous hemagglutinin protein of Bordetella pertussis. Monoclonal antibody X3C, a murine immunoglobulin G (IgG) antibody which recognizes filamentous hemagglutinin, was used to probe cell sonicates by Western blot. An alkaline phosphatase-conjugated goat anti-mouse IgG second antibody was used for detection.
To generate recombinant protein antiserum, E. coli BL21(DE3)/pLysS was transformed with pHMW1-4, and expression of recombinant protein was induced with IPTG, as described above. A cell sonicate of the bacterial cells was prepared and separated into a supernatant and pellet fraction by centrifugation at 10,000.times.g for 30 min. The recombinant protein fractionated with the pellet fraction. A rabbit was subcutaneously immunized on biweekly schedule with 1 mg of protein from the pellet fraction, the first dose given with Freund's complete adjuvant and subsequent doses with Freund's incomplete adjuvant. Following the fourth injection, the rabbit was bled. Prior to use in the Western blot assay, the antiserum was absorbed extensively with sonicates of the host E. coli strain transformed with cloning vector alone.
To assess the sharing of antigenic determinants between HMW1 and filamentous hemagglutinin, enzyme-linked immunosorbent assay (ELISA) plates (Costar, Cambridge, Mass.) were coated with 60 .mu.l of a 4-ug/ml solution of filamentous hemagglutinin in Dulbecco's phosphate-buffered saline per well for 2 h at room temperature. Wells were blocked for 1 h with 1% bovine serum albumin in Dulbecco's phosphate-buffered saline prior to addition of serum dilutions. rHMW1 antiserum was serially diluted in 0.1% Brij (Sigma, St. Louis, Mo.) in Dulbecco's phosphate-buffered saline and incubated for 3 h at room temperature. After being washed, the plates were incubated with peroxidase-conjugated goat anti-rabbit lgG antibody (Bio-Rad) for 2 h at room temperature and subsequently developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) at a concentration of 0.54 in mg/ml in 0.1M sodium citrate buffer, pH 4.2, containing 0.03% H.sub.2 O.sub.2. Absorbances were read on an automated ELISA reader.
Recombinant phage expressing HMW1 or HMW2 were recovered as follows. The non-typeable H. influenzae strain 12 genomic library was screened for clones expressing high-molecular-weight proteins with an E. coli-absorbed human serum sample containing a high titer of antibodies directed against the high-molecular-weight proteins.
Numerous strongly reactive clones were identified along with more weakly reactive ones. Twenty strongly reactive clones were plaque-purified and examined by Western blot for expression of recombinant proteins. Each of the strongly reactive clones expressed one of two types of high-molecular-weight proteins, designated HMW1 and HMW2. The major immunoreactive protein bands in the HMW1 and HMW2 lysates migrated with apparent molecular masses of 125 and 120 kDa, respectively. In addition to the major bands, each lysate contained minor protein bands of higher apparent molecular weight. Protein bands seen in the HMW2 lysates at molecular masses of less than 120 kDa were not regularly observed and presumably represent proteolytic degradation products. Lysates of LE392 infected with the .lambda.EMBL3 cloning vector alone were non-reactive when immunologically screened with the same serum sample. Thus, the observed activity was not due to cross-reactive E. coli proteins or .lambda.EMBL3-encoded proteins. Furthermore, the recombinant proteins were not simply binding immunoglobulin nonspecifically, since the proteins were not reactive with the goat anti-human IgG conjugate alone, with normal rabbit sera, or with serum from a number of healthy young infants.
Representative clones expressing either the HMW1 or HMW2 recombinant proteins were characterized further. The restriction maps of the two phage types were different from each other, including the regions encoding the HMW1 and HMW2 structural genes. FIG. 5A shows restriction maps of representative recombinant phage which contained the HMW1 or HMW2 structural genes. The locations of the structural genes are indicated by the shaded bars.
HMW1 plasmid subclones were constructed by using the T7 expression plasmid T7-7 (FIG. 5A and B). HMW2 plasmid subclones also were constructed, and the results with these latter subclones were similar to those observed with the HMW1 constructs.
The approximate location and direction of transcription of the HMW1 structure gene were initially determined by using plasmid pHMW1 (FIG. 5A). This plasmid was constructed by inserting the 8.5-kb BamHI-SalI fragment from .lambda.HMW1 into BamHI- and SalI-cut pT7-7. E. Coli transformed with pHMW1 expressed an immunoreactive recombinant protein with an apparent molecular mass of 115 kDa, which was strongly inducible with IPTG. This protein was significantly smaller than the 125-kDa major protein expressed by the parent phage, indicating that it either was being expressed as a fusion protein or was truncated at the carboxy terminus.
To more precisely localize the 3' end of the structural gene, additional plasmids were constructed with progressive deletions from the 3' end of the pHMW1 construct. Plasmid pHMW1-1 was constructed by digestion of pHMW1 with PstI, isolation of the resulting 8.8-kb fragment, and religation. Plasmid pHMW1-2 was constructed by digestion of pHMW1 with HindIII, isolation of the resulting 7.5-kb fragment, and religation. E. coli transformed with either plasmid pHMW1-1 or pHMW1-2 also expressed an immunoreactive recombinant protein with an apparent molecular mass of 115 kDa. These results indicated that the 3' end of the structural gene was 5' of the HindIII site.
To more precisely localize the 5' end of the gene, plasmids pHMW1-4 and pHMW1-7 were constructed. Plasmid pHMW1-4 was constructed by cloning the 5.1-kb BamHI-HindIII fragment from .lambda.HMW1 into a pT7-7-derived plasmid containing the upstream 3.8-kb EcoRI-BamHi fragment. E. coli transformed with pHMW1-4 expressed an immunoreactive protein with an apparent molecular mass of approximately 160 kDa. Although protein production was inducible with IPTG, the levels of protein production in these transformants were substantially lower than those with the pHMW1-2 transformants described above. Plasmid pHMW1-7 was constructed by digesting pHMW1-4 with NdeI and SpeI. The 9.0-kbp fragment generated by this double digestion was isolated, blunt ended, and religated. E. coli transformed with pHMW1-7 also expressed an immunoreactive protein with an apparent molecular mass of 160 kDa, a protein identical in size to that expressed by the pHMW1-4 transformants. The result indicated that the initiation codon for the HMW1 structural gene was 3' of the SpeI site. DNA sequence analysis confirmed this conclusion.
As noted above, the .lambda.HMW1 phage clones expressed a major immunoreactive band of 125 kDa, whereas the HMW1 plasmid clones pHMW1-4 and pHMW1-7, which contained what was believed to be the full-length gene, expressed an immunoreactive protein of approximately 160 kDa. This size discrepancy was disconcerting. One possible explanation was that an additional gene or genes necessary for correct processing of the HMW1 gene product were deleted in the process of subcloning. To address this possibility, plasmid pHMW1-14 was constructed. This construct was generated by digesting pHMW1 with NdeI and MluI and inserting the 7.6-kbp NdeI-MluI fragment isolated from pHMW1-4. Such a construct would contain the full-length HMW1 gene as well as the DNA 3' of the HMW1 gene which was present in the original HMW1 phage. E. coli transformed with this plasmid expressed major immunoreactive proteins with apparent molecular masses of 125 and 160 kDa as well as additional degradation products. The 125- and 160-kDa bands were identical to the major and minor immunoreactive bands detected in the HMW1 phage lysates. Interestingly, the pHMW1-14 construct also expressed significant amounts of protein in the uninduced condition, a situation not observed with the earlier constructs.
The relationship between the 125- and 160-kDa proteins remains somewhat unclear. Sequence analysis, described below, reveals that the HMW1 gene would be predicted to encode a protein of 159 kDa. It is believed that the 160-kDa protein is a precursor form of the mature 125-kDa protein, with the conversion from one protein to the other being dependent on the products of the two downstream genes.
Sequence analysis of the HMW1 gene (FIG. 1) revealed a 4,608-bp open reading frame (ORF), beginning with an ATG codon at nucleotide 351 and ending with a TAG stop codon at nucleotide 4959. A putative ribosome-binding site with the sequence AGGAG begins 10 bp up-stream of the putative initiation codon. Five other in-frame ATG codons are located within 250 bp of the beginning of the ORF, but none of these is preceded by a typical ribosome-binding site. The 5'-flanking region of the ORF contains a series of direct tandem repeats, with the 7-bp sequence ATCTTTC repeated 16 times. These tandem repeats stop 100 bp 5' of the putative initiation codon. An 8-bp inverted repeat characteristic of a rho-independent transcriptional terminator is present, beginning at nucleotide 4983, 25 bp 3' of the presumed translational stop. Multiple termination codons are present in all three reading frames both upstream and downstream of the ORF. The derived amino acid sequence of the protein encoded by the HMW1 gene (FIG. 2) has a molecular weight of 159,000, in good agreement with the apparent molecular weights of the proteins expressed by the HMW1-4 and HMW1-7 transformants. The derived amino acid sequence of the amino terminus does not demonstrate the characteristics of a typical signal sequence. The BamHI site used in generation of pHMW1 comprises bp 1743 through 1748 of the nucleotide sequence. The ORF downstream of the BamHI site would be predicted to encode a protein of 111 kDa, in good agreement with the 115 kDa estimated for the apparent molecular mass of the pHMW1-encoded fusion protein.
The sequence of the HMW2 gene (FIG. 3) consists of a 4,431-bp ORF, beginning with an ATG codon at nucleotide 352 and ending with a TAG stop codon at nucleotide 4783. The first 1,259 bp of the ORF of the HMW2 gene are identical to those of the HMW1 gene. Thereafter, the sequences begin to diverge but are 80% identical overall. With the exception of a single base addition at nucleotide 93 of the HMW2 sequence, the 5'-flanking regions of the HMW1 and HMW2 genes are identical for 310 bp upstream from the respective initiation codons. Thus, the HMW2 gene is preceded by the same set of tandem repeats and the same putative ribosome-binding site which lies 5' of the HMW1 gene. A putative transcriptional terminator identical to that identified 3' of the HMW1 ORF is noted, beginning at nucleotide 4804. The discrepancy in the lengths of the two genes is principally accounted for by a 186-bp gap in the HMW2 sequence, beginning at nucleotide position 3839. The derived amino acid sequence of the protein encoded by the HMW2 gene (FIG. 4) has a molecular weight of 155,000 and is 71% identical with the derived amino acid sequence of the HMW1 gene.
The derived amino acid sequences of both the HMW1 and HMW2 genes (FIGS. 2 and 4) demonstrated sequence similarity with the derived amino acid sequence of filamentous hemagglutinin of Bordetella pertussis, a surface-associated protein of this organism. The initial and optimized TFASTA scores for the HMW1-filamentous hemagglutinin sequence comparison were 87 and 186, respectively, with a word size of 2. The z score for the comparison was 45.8. The initial and optimized TFASTA scores for the HMW2-filamentous hemagglutinin sequence comparison were 68 and 196, respectively. The z score for the latter comparison was 48.7. The magnitudes of the initial and optimized TFASTA scores and the z scores suggested that a biologically significant relationship existed between the HMW1 and HMW2 gene products and filamentous hemagglutinin. When the derived amino acid sequences of HMW1, HMW2, and filamentous hemagglutinin genes were aligned and compared, the similarities were most notable at the amino-terminal ends of the three sequences. Twelve of the first 22 amino acids in the predicted peptide sequences were identical. In additional, the sequences demonstrated a common five-amino-acid stretch, Asn-Pro-Asn-Gly-Ile, and several shorter stretches of sequence identity within the first 200 amino acids.
Example 2To further explore the HMW1-filamentous hemagglutinin relationship, the ability of antiserum prepared against the HMW1-4 recombinant protein (rHMW1) to recognize purified filamentous hemagglutinin was assessed. The rHMW1 antiserum demonstrated ELISA reactivity with filamentous hemagglutinin in a dose-dependent manner. Preimmune rabbit serum had minimal reactivity in this assay. The rHMW1 antiserum also was examined in a Western blot assay and demonstrated weak but positive reactivity with purified filamentous hemagglutinin in this system also.
To identify the native Haemophilus protein corresponding to the HMW1 gene product and to determine the extent to which proteins antigenically related to the HMW1 cloned gene product were common among other non-typeable H. influenzae strains, a panel of Haemophilus strains was screened by Western blot with the rHMW1 antiserum. The antiserum recognized both a 125- and a 120-kDa protein band in the homologous strain 12, the putative mature protein products of the HMW1 and HMW2 genes, respectively.
When used to screen heterologous non-typeable H. influenzae strains, rHMW1 antiserum recognized high-molecular-weight proteins in 75% of 125 epidemiologically unrelated strains. In general, the antiserum reacted with one or two protein bands in the 100- to 150-kDa range in each of the heterologous strains in a pattern similar but not identical to that seen in the homologous strain.
Monoclonal antibody X3C is a murine IgG antibody directed against the filamentous hemagglutinin protein of B. pertussis. This antibody can inhibit the binding of B. pertussis cells to Chinese hamster ovary cells and HeLa cells in culture and will inhibit hemagglutination of erythrocytes by purified filamentous hemagglutinin. A Western blot assay was performed in which this monoclonal antibody was screened against the same panel of non-typeable H. influenzae strains discussed above. Monoclonal antibody X3C recognized both the high-molecular-weight proteins in non-typeable H. influenzae strain 12 which were recognized by the recombinant-protein antiserum. In addition, the monoclonal antibody recognized protein bands in a subset of heterologous non-typeable H. influenzae strains which were identical to those recognized by the recombinant-protein antiserum. On occasion, the filamentous hemagglutinin monoclonal antibody appeared to recognize only one of the two bands which had been recognized by the recombinant-protein antiserum. Overall, monoclonal antibody X3C recognized high-molecular-weight protein bands identical to those recognized by the rHMW1 antiserum in approximately 35% of our collection of non-typeable H. influenzae strains.
Example 3Mutants deficient in expression of HMW1, MW2 or both proteins were constructed to examine the role of these proteins in bacterial adherence. The following strategy was employed. pHMW1-14 (see Example 1, FIG. 5A) was digested with BamHI and then ligated to a kanamycin cassette isolated on a 1.3-kb BamHl fragment from pUC4K. The resultant plasmid (pHMW1-17) was linearized by digestion with XbaI and transformed into non-typeable H. influenzae strain 12, followed by selection for kanamycin resistant colonies. Southern analysis of a series of these colonies demonstrated two populations of transformants, one with an insertion in the HMW1 structural gene and the other with an insertion in the HMW2 structural gene. One mutant from each of these classes was selected for further studies.
Mutants deficient in expression of both proteins were recovered using the following protocol. After deletion of the 2.1-kb fragment of DNA between two EcoRI sites spanning the 3'-portion of the HMW1 structural gene in pHMW-15, the kanamycin cassette from pUC4K was inserted as a 1.3-kb EcoRl fragment. The resulting plasmid (pHMW1-16) was linearized by digestion with XbaI and transformed into strain 12, followed again by selection for kanamycin resistant colonies. Southern analysis of a representative sampling of these colonies demonstrated that in seven of eight cases, insertion into both the HMW1 and HMW2 loci had occurred. One such mutant was selected for further studies.
To confirm the intended phenotypes, the mutant strains were examined by Western blot analysis with a polyclonal antiserum against recombinant HMW1 protein. The parental strain expressed both the 125-kD HMW1 and the 120-kD HMW2 protein. In contrast, the HMW2 mutant failed to express the 120-kD protein, and the HMW1 mutant failed to express the 125-kD protein. The double mutant lacked expression of either protein. On the basis of whole cell lysates, outer membrane profiles, and colony morphology, the wild type strain and the mutants were otherwise identical with one another. Transmission electron microscopy demonstrated that none of the four strains expressed pili.
The capacity of wild type strain 12 to adhere to Chang epithelial cells was examined. In such assays, bacteria were inoculated into broth and allowed to grow to a density of .about.2.times.10.sup.9 cfu/ml. Approximately 2.times.10.sup.7 cfu were inoculated onto epithelial cell monolayers, and plates were gently centrifuged at 165.times.g for 5 minutes to facilitate contact between bacteria and the epithelial surface. After incubation for 30 minutes at 37.degree. C. in 5% CO.sub.2, monolayers were rinsed 5 times with PBS to remove nonadherent organisms and were treated with trypsin-EDTA (0.05% trypsin, 0.5% EDTA) in PBS to release them from the plastic support. Well contents were agitated, and dilutions were plated on solid medium to yield the number of adherent bacteria per monolayer. Percent adherence was calculated by dividing the number of adherent cfu per monolayer by the number of inoculated cfu.
As depicted in Table 1 below (the Tables appear at the end of the descriptive text), this strain adhered quite efficiently, with nearly 90% of the inoculum binding to the monolayer. Adherence by the mutant expressing HMW1 but not HMW2 (HMW2.sup.-) was also quite efficient and comparable to that by the wild type strain. In contrast, attachment by the strain expressing HMW2 but deficient in expression of HMW1 (HMW1.sup.-) was decreased about 15-fold relative to the wild type. Adherence by the double mutant (HMW1.sup.- /HMW2.sup.-) was decreased even further, approximately 50-fold compared with the wild type and approximately 3-fold compared with the HMW1 mutant. Considered together, these results suggest that both the HMW1 protein and the, HMW2 protein influence attachment to Chang epithelial cells. Interestingly, optimal adherence to this cell line appears to require HMW1 but not HMW2.
Example 4Using the plasmids pHMW1-16 and pHMW1-17 (see Example 3) and following a scheme similar to that employed with strain 12 as described in Example 3, three non-typeable Haemophilus strain 5 mutants were isolated, including one with the kanamycin gene inserted into the hmw1-like (designated hmw3) locus, a second with an insertion in the hmw2-like (designated hmw4) locus, and a third with insertions in both loci. As predicted, Western immunoblot analysis demonstrated that the mutant with insertion of the kanamycin cassette into the hmw1-like locus had lost expression of the HMW3 125-kD protein, while the mutant with insertion into the hmw2-like locus failed to express the HMW4 123-kD protein. The mutant with a double insertion was unable to express either of the high molecular weight proteins.
As shown in Table 1 below, wild type strain 5 demonstrated high level adherence, with almost 80% of the inoculum adhering per monolayer. Adherence by the mutant deficient in expression of the HMW2-like protein was also quite high. In contrast, adherence by the mutant unable to express the, HMW1-like protein was reduced about 5-fold relative to the wild type, and attachment by the double mutant was diminished even further (approximately 25-fold). Examination of Giemsa-stained samples confirmed these observations (not shown). Thus, the results with strain 5 corroborate the findings with strain 12 and the HMW1 and HMW2 proteins.
Example 5To confirm an adherence function for the HMW1 and HMW2 proteins and to examine the effect of HMW1 and HMW2 independently of other H. influenzae surface structures, the hmw1 and the hmw2 gene clusters were introduced into E. coli DH5.alpha., using plasmids pHMW1-14 and pHMW2-21, respectively. As a control, the cloning vector, pT7-7, was also transformed into E. coli DH5.alpha.. Western blot analysis demonstrated that E. coli DH5.alpha. containing the hmw1 genes expressed a 125 kDa protein, while the same strain harboring the hmw2 genes expressed a 120-kDa protein. E. coli DH5.alpha. containing pT7-7 failed to react with antiserum against recombinant HMW1. Transmission electron microscopy revealed no pili or other surface appendages on any of the E. coli strains.
Adherence by the E. coli strains was quantitated and compared with adherence by wild type non-typeable H. influenzae strain 12. As shown in Table 2 below, adherence by E. coli DH5.alpha. containing vector alone was less than 1% of that for strain 12. In contrast, E. coli DH5.alpha. harboring the hmw1 gene cluster demonstrated adherence levels comparable to those for strain 12. Adherence by E. coli DH5.alpha. containing the hmw2 genes was approximately 6-fold lower than attachment by strain 12 but was increased 20-fold over adherence by E. coli DH5.alpha. with pT7-7 alone. These results indicate that the HMW1 and HMW2 proteins are capable of independently mediating attachment to Chang conjunctival cells. These results are consistent with the results with the H. influenzae mutants reported in Examples 3 and 4, providing further evidence that, with Chang epithelial cells, HMW1 is a more efficient adhesin than is HMW2.
Experiments with E. coli HB101 harboring pT7-7, pHMW1-14, or pHMW2-21 confirmed the results obtained with the DH5.alpha. derivatives (see Table 2).
Example 6HMW1 and HMW2 were isolated and purified from non-typeable H. influenzae (NTHI) strain 12 in the following manner. Non-typeable Haemophilus bacteria from frozen stock culture were streaked onto a chocolate plate and grown overnight at 37.degree. C. in an incubator with 5% CO.sub.2. 50 ml starter culture of brain heart infusion (BHI) broth, supplemented with 10 .mu.g/ml each of hemin and NAD was inoculated with growth on chocolate plate. The starter culture was grown until the optical density (O.D.--600 nm) reached 0.6 to 0.8 and then the bacteria in the starter culture was used to inoculate six 500 ml flasks of supplemented BHI using 8 to 10 ml per flask. The bacteria were grown in 500 ml flasks for an additional 5 to 6 hours at which time the O.D. was 1.5 or greater. Cultures were centrifuged at 10,000 rpm for 10 minutes.
Bacterial pellets were resuspended in a total volume of 250 ml of an extraction solution comprising 0.5M NaCl, 0.01M Na.sub.2 EDTA, 0.01M Tris 50 .mu.M 1,10-phenanthroline, pH 7.5. The cells were not sonicated or otherwise disrupted. The resuspended cells were allowed to sit on ice at 0.degree. C. for 60 minutes. The resuspended cells were centrifuged at 10,000 rpm for 10 minutes at 4.degree. C. to remove the majority of intact cells and cellular debris. The supernatant was collected and centrifuged at 100,000.times.g for 60 minutes at 4.degree. C. The supernatant again was collected and dialyzed overnight at 4.degree. C. against 0.01M sodium phosphate, pH 6.0.
The sample was centrifuged at 10,000 rpm for 10 minutes at 4.degree. C. to remove insoluble debris precipitated from solution during dialysis. The supernatant was applied to a 10 ml CM Sepharose column which has been pre-equilibrated with 0.01M sodium phosphate, pH 6. Following application to this column, the column was washed with 0.01M sodium phosphate. Proteins were elevated from the column with a 0-0.5M KCl gradient in 0.01M Na phosphate, pH 6 and fractions were collected for gel examination. Coomassie gels of column fractions were carried out to identify those fractions containing high molecular weight proteins. The fractions containing high molecular weight proteins were pooled and concentrated to a 1 to 3 ml volume in preparation for application of sample to gel filtration column.
A Sepharose CL-4B gel filtration column was equilibrated with phosphate-buffered saline, pH 7.5. The concentrated high molecular weight protein sample was applied to the gel filtration column and column fractions were collected. Coomassie gels were performed on the column fractions to identify those containing high molecular weight proteins. The column fractions containing high molecular weight proteins were pooled.
The proteins were tested to determine whether they would protect against experimental otitis media caused by the homologous strain.
Chinchillas received three monthly subcutaneous injections with 40 .mu.g of an HMW1-HMW2 protein mixture in Freund's adjuvant. One month after the last injection, the animals were challanged by intrabullar inoculation with 300 cfu of NTHI strain 12.
Infection developed in 5 of 5 control animals versus 5 of 10 immunized animals. Among infected animals, geometric mean bacterial counts in middle ear fluid 7 days post-challenge were 7.4.times.10.sup.6 in control animals versus 1.3.times.10.sup.5 in immunized animals.
Serum antibody titres following immunization were comparable in uninfected and infected animals. However, infection in immunized animals was uniformly associated with the appearance of bacteria down-regulated in expression of the HMW proteins, suggesting bacterial selection in response to immunologic pressure.
Although this data shows that protection following immunization was not complete, this data suggests the HMW adhesin proteins are potentially important protective antigens which may comprise one component of a multi-component NTHI vaccine.
Example 7A number of synthetic peptides were derived from HMW1. Antisera then was raised to these peptides. The anti-peptide antisera to peptide HMW1-P5 was shown to recognize HMW1. Peptide HMW1-P5 covers amino acids 1453 to 1481 of HMW1, has the sequence VDEVIEAKRILEKVKDLSDEEREALAKLG (SEQ ID NO:9), and represents bases 1498 to 1576 in FIG. 10.
This finding demonstrates that the DNA sequence and the derived protein is being interpreted in the correct reading frame and that peptides derived from the sequence can be produced which will be immunogenic.
SUMMARY OF DISCLOSUREIn summary of this disclosure, the present invention provides high molecular weight proteins of non-typeable Haemophilus, genes coding for the same and vaccines incorporating such proteins. Modifications are possible within the scope of this invention.
TABLE 1
______________________________________
Effect of mutation of high molecular weight
proteins on adherence to Chang epithelial cells by
nontypable H. influenzae.
ADHERENCE*
Strain % inoculum relative to wild type.dagger.
______________________________________
Strain 12 derivatives
wild type 87.7 .+-. 5.9
100.0 .+-. 6.7
HMW1-mutant 6.0 .+-. 0.9
6.8 .+-. 1.0
HMW2-mutant 89.9 .+-. 10.8
102.5 .+-. 12.3
HMW1-/HMW2-mutant
2.0 .+-. 0.3
2.3 .+-. 0.3
Strain 5 derivatives
wild type 78.7 .+-. 3.2
100.0 .+-. 4.1
HMW1-like mutant
15.7 .+-. 2.6
19.9 .+-. 3.3
HMW2-like mutant
103.7 .+-. 14.0
131.7 .+-. 17.8
double mutant 3.5 .+-. 0.6
4.4 .+-. 0.8
______________________________________
*Numbers represent mean (.+-. standard error of the mean) of measurements
in triplicate or quadruplicate from representative experiments.
.dagger.Adherence values for strain 12 derivatives are relative to strain
12 wild type; values for strain 5 derivatives are relative to strain 5
wild type.
TABLE 2
______________________________________
Adherence by E. coli DH5.alpha. and HB101 harboring
hmw1 or hmw2 gene clusters.
Adherence relative to
Strain* H. influenzae strain 12.dagger.
______________________________________
DH5.alpha. (pT7-7)
0.7 .+-. 0.02
DH5.alpha. (pHMW1-14)
114.2 .+-. 15.9
DH5.alpha. (pHMW2-21)
14.0 .+-. 3.7
HB101 (pT7-7) 1.2 .+-. 0.5
HB101 (pHMW1-14) 93.6 .+-. 15.8
HB101 (pHMW2-21) 3.6 .+-. 0.9
______________________________________
*The plasmid pHMW114 contains the hmw1 gene cluster, while pHMW221
contains the hmw2 gene cluster; pT77 is the cloning vector used in these
constructs.
.dagger.Numbers represent the mean (.+-. standard error of the mean) of
measurements made in triplicate from representative experiments.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 8
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5116 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ACAGCGTTCTCTTAATACTAGTACAAACCCACAATAAAATATGACAAACAACAATTACAA60
CACCTTTTTTGCAGTCTATATGCAAATATTTTAAAAAATAGTATAAATCCGCCATATAAA120
ATGGTATAATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATC180
TTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTC240
ACATGCCCTGATGAACCGAGGGAAGGGAGGGAGGGGCAAGAATGAAGAGGGAGCTGAACG300
AACGCAAATGATAAAGTAATTTAATTGTTCAACTAACCTTAGGAGAAAATATGAACAAGC360
TATATCGTCTCAAATTCAGCAAACGCCTGAATGCTTTGGTTGCTGTGTCTGAATTGGCAC420
GGGGTTGTGACCATTCCACAGAAAAAGGCAGCGAAAAACCTGCTCGCATGAAAGTGCGTC480
ACTTAGCGTTAAAGCCACTTTCCGCTATGTTACTATCTTTAGGTGTAACATCTATTCCAC540
AATCTGTTTTAGCAAGCGGCTTACAAGGAATGGATGTAGTACACGGCACAGCCACTATGC600
AAGTAGATGGTAATAAAACCATTATCCGCAACAGTGTTGACGATATCATTAATTGGAAAC660
AATTTAACATCGACCAAAATGAAATGGTGCAGTTTTTACAAGAAAACAACAACTCCGCCG720
TATTCAACCGTGTTACATCTAACCAAATCTCCCAATTAAAAGGGATTTTAGATTCTAACG780
GACAAGTCTTTTTAATCAACCCAAATGGTATCACAATAGGTAAAGACGCAATTATTAACA840
CTAATGGCTTTACGGCTTCTACGCTAGACATTTCTAACGAAAACATCAAGGCGCGTAATT900
TCACCTTCGAGCAAACCAAAGATAAAGCGCTCGCTGAAATTGTGAATCACGGTTTAATTA960
CTGTCGGTAAAGACGGCAGTGTAAATCTTATTGGTGGCAAAGTGAAAAACGAGGGTGTGA1020
TTAGCGTAAATGGTGGCAGCATTTCTTTACTCGCAGGGCAAAAAATCACCATCAGCGATA1080
TAATAAACCCAACCATTACTTACAGCATTGCCGCGCCTGAAAATGAAGCGGTCAATCTGG1140
GCGATATTTTTGCCAAAGGCGGTAACATTAATGTCCGTGCTGCCACTATTCGAAACCAAG1200
GTAAACTTTCTGCTGATTCTGTAAGCAAAGATAAAAGCGGCAATATTGTTCTTTCCGCCA1260
AAGAGGGTGAAGCGGAAATTGGCGGTGTAATTTCCGCTCAAAATCAGCAAGCTAAAGGCG1320
GCAAGCTGATGATTACAGGCGATAAAGTCACATTAAAAACAGGTGCAGTTATCGACCTTT1380
CAGGTAAAGAAGGGGGAGAAACTTACCTTGGCGGTGACGAGCGCGGCGAAGGTAAAAAGG1440
GCATTCAATTAGCAAAGAAAACCTCTTTAGAAAAAGGCTCAACCATCAATGTATCAGGCA1500
AAGAAAAAGGCGGACGCGCTATTGTGTGGGGCGATATTGCGTTAATTGACGGCAATATTA1560
ACGCTCAAGGTAGTGGTGATATCGCTAAAACCGGTGGTTTTGTGGAGACGTCGGGGCATG1620
ATTTATTCATCAAAGACAATGCAATTGTTGACGCCAAAGAGTGGTTGTTAGACCCGGATA1680
ATGTATCTATTAATGCAGAAACAGCAGGACGCAGCAATACTTCAGAAGACGATGAATACA1740
CGGGATCCGGGAATAGTGCCAGCACCCCAAAACGAAACAAAGAAAAGACAACATTAACAA1800
ACACAACTCTTGAGAGTATACTAAAAAAAGGTACCTTTGTTAACATCACTGCTAATCAAC1860
GCATCTATGTCAATAGCTCCATTAATTTATCCAATGGCAGCTTAACTCTTTGGAGTGAGG1920
GTCGGAGCGGTGGCGGCGTTGAGATTAACAACGATATTACCACCGGTGATGATACCAGAG1980
GTGCAAACTTAACAATTTACTCAGGCGGCTGGGTTGATGTTCATAAAAATATCTCACTCG2040
GGGCGCAAGGTAACATAAACATTACAGCTAAACAAGATATCGCCTTTGAGAAAGGAAGCA2100
ACCAAGTCATTACAGGTCAAGGGACTATTACCTCAGGCAATCAAAAAGGTTTTAGATTTA2160
ATAATGTCTCTCTAAACGGCACTGGCAGCGGACTGCAATTCACCACTAAAAGAACCAATA2220
AATACGCTATCACAAATAAATTTGAAGGGACTTTAAATATTTCAGGGAAAGTGAACATCT2280
CAATGGTTTTACCTAAAAATGAAAGTGGATATGATAAATTCAAAGGACGCACTTACTGGA2340
ATTTAACCTCCTTAAATGTTTCCGAGAGTGGCGAGTTTAACCTCACTATTGACTCCAGAG2400
GAAGCGATAGTGCAGGCACACTTACCCAGCCTTATAATTTAAACGGTATATCATTCAACA2460
AAGACACTACCTTTAATGTTGAACGAAATGCAAGAGTCAACTTTGACATCAAGGCACCAA2520
TAGGGATAAATAAGTATTCTAGTTTGAATTACGCATCATTTAATGGAAACATTTCAGTTT2580
CGGGAGGGGGGAGTGTTGATTTCACACTTCTCGCCTCATCCTCTAACGTCCAAACCCCCG2640
GTGTAGTTATAAATTCTAAATACTTTAATGTTTCAACAGGGTCAAGTTTAAGATTTAAAA2700
CTTCAGGCTCAACAAAAACTGGCTTCTCAATAGAGAAAGATTTAACTTTAAATGCCACCG2760
GAGGCAACATAACACTTTTGCAAGTTGAAGGCACCGATGGAATGATTGGTAAAGGCATTG2820
TAGCCAAAAAAAACATAACCTTTGAAGGAGGTAACATCACCTTTGGCTCCAGGAAAGCCG2880
TAACAGAAATCGAAGGCAATGTTACTATCAATAACAACGCTAACGTCACTCTTATCGGTT2940
CGGATTTTGACAACCATCAAAAACCTTTAACTATTAAAAAAGATGTCATCATTAATAGCG3000
GCAACCTTACCGCTGGAGGCAATATTGTCAATATAGCCGGAAATCTTACCGTTGAAAGTA3060
ACGCTAATTTCAAAGCTATCACAAATTTCACTTTTAATGTAGGCGGCTTGTTTGACAACA3120
AAGGCAATTCAAATATTTCCATTGCCAAAGGAGGGGCTCGCTTTAAAGACATTGATAATT3180
CCAAGAATTTAAGCATCACCACCAACTCCAGCTCCACTTACCGCACTATTATAAGCGGCA3240
ATATAACCAATAAAAACGGTGATTTAAATATTACGAACGAAGGTAGTGATACTGAAATGC3300
AAATTGGCGGCGATGTCTCGCAAAAAGAAGGTAATCTCACGATTTCTTCTGACAAAATCA3360
ATATTACCAAACAGATAACAATCAAGGCAGGTGTTGATGGGGAGAATTCCGATTCAGACG3420
CGACAAACAATGCCAATCTAACCATTAAAACCAAAGAATTGAAATTAACGCAAGACCTAA3480
ATATTTCAGGTTTCAATAAAGCAGAGATTACAGCTAAAGATGGTAGTGATTTAACTATTG3540
GTAACACCAATAGTGCTGATGGTACTAATGCCAAAAAAGTAACCTTTAACCAGGTTAAAG3600
ATTCAAAAATCTCTGCTGACGGTCACAAGGTGACACTACACAGCAAAGTGGAAACATCCG3660
GTAGTAATAACAACACTGAAGATAGCAGTGACAATAATGCCGGCTTAACTATCGATGCAA3720
AAAATGTAACAGTAAACAACAATATTACTTCTCACAAAGCAGTGAGCATCTCTGCGACAA3780
GTGGAGAAATTACCACTAAAACAGGTACAACCATTAACGCAACCACTGGTAACGTGGAGA3840
TAACCGCTCAAACAGGTAGTATCCTAGGTGGAATTGAGTCCAGCTCTGGCTCTGTAACAC3900
TTACTGCAACCGAGGGCGCTCTTGCTGTAAGCAATATTTCGGGCAACACCGTTACTGTTA3960
CTGCAAATAGCGGTGCATTAACCACTTTGGCAGGCTCTACAATTAAAGGAACCGAGAGTG4020
TAACCACTTCAAGTCAATCAGGCGATATCGGCGGTACGATTTCTGGTGGCACAGTAGAGG4080
TTAAAGCAACCGAAAGTTTAACCACTCAATCCAATTCAAAAATTAAAGCAACAACAGGCG4140
AGGCTAACGTAACAAGTGCAACAGGTACAATTGGTGGTACGATTTCCGGTAATACGGTAA4200
ATGTTACGGCAAACGCTGGCGATTTAACAGTTGGGAATGGCGCAGAAATTAATGCGACAG4260
AAGGAGCTGCAACCTTAACTACATCATCGGGCAAATTAACTACCGAAGCTAGTTCACACA4320
TTACTTCAGCCAAGGGTCAGGTAAATCTTTCAGCTCAGGATGGTAGCGTTGCAGGAAGTA4380
TTAATGCCGCCAATGTGACACTAAATACTACAGGCACTTTAACTACCGTGAAGGGTTCAA4440
ACATTAATGCAACCAGCGGTACCTTGGTTATTAACGCAAAAGACGCTGAGCTAAATGGCG4500
CAGCATTGGGTAACCACACAGTGGTAAATGCAACCAACGCAAATGGCTCCGGCAGCGTAA4560
TCGCGACAACCTCAAGCAGAGTGAACATCACTGGGGATTTAATCACAATAAATGGATTAA4620
ATATCATTTCAAAAAACGGTATAAACACCGTACTGTTAAAAGGCGTTAAAATTGATGTGA4680
AATACATTCAACCGGGTATAGCAAGCGTAGATGAAGTAATTGAAGCGAAACGCATCCTTG4740
AGAAGGTAAAAGATTTATCTGATGAAGAAAGAGAAGCGTTAGCTAAACTTGGAGTAAGTG4800
CTGTACGTTTTATTGAGCCAAATAATACAATTACAGTCGATACACAAAATGAATTTGCAA4860
CCAGACCATTAAGTCGAATAGTGATTTCTGAAGGCAGGGCGTGTTTCTCAAACAGTGATG4920
GCGCGACGGTGTGCGTTAATATCGCTGATAACGGGCGGTAGCGGTCAGTAATTGACAAGG4980
TAGATTTCATCCTGCAATGAAGTCATTTTATTTTCGTATTATTTACTGTGTGGGTTAAAG5040
TTCAGTACGGGCTTTACCCATCTTGTAAAAAATTACGGAGAATACAATAAAGTATTTTTA5100
ACAGGTTATTATTATG5116
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1536 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetAsnLysIleTyrArgLeuLysPheSerLysArgLeuAsnAlaLeu
151015
ValAlaValSerGluLeuAlaArgGlyCysAspHisSerThrGluLys
202530
GlySerGluLysProAlaArgMetLysValArgHisLeuAlaLeuLys
354045
ProLeuSerAlaMetLeuLeuSerLeuGlyValThrSerIleProGln
505560
SerValLeuAlaSerGlyLeuGlnGlyMetAspValValHisGlyThr
65707580
AlaThrMetGlnValAspGlyAsnLysThrIleIleArgAsnSerVal
859095
AspAlaIleIleAsnTrpLysGlnPheAsnIleAspGlnAsnGluMet
100105110
ValGlnPheLeuGlnGluAsnAsnAsnSerAlaValPheAsnArgVal
115120125
ThrSerAsnGlnIleSerGlnLeuLysGlyIleLeuAspSerAsnGly
130135140
GlnValPheLeuIleAsnProAsnGlyIleThrIleGlyLysAspAla
145150155160
IleIleAsnThrAsnGlyPheThrAlaSerThrLeuAspIleSerAsn
165170175
GluAsnIleLysAlaArgAsnPheThrPheGluGlnThrLysAspLys
180185190
AlaLeuAlaGluIleValAsnHisGlyLeuIleThrValGlyLysAsp
195200205
GlySerValAsnLeuIleGlyGlyLysValLysAsnGluGlyValIle
210215220
SerValAsnGlyGlySerIleSerLeuLeuAlaGlyGlnLysIleThr
225230235240
IleSerAspIleIleAsnProThrIleThrTyrSerIleAlaAlaPro
245250255
GluAsnGluAlaValAsnLeuGlyAspIlePheAlaLysGlyGlyAsn
260265270
IleAsnValArgAlaAlaThrIleArgAsnGlnGlyLysLeuSerAla
275280285
AspSerValSerLysAspLysSerGlyAsnIleValLeuSerAlaLys
290295300
GluGlyGluAlaGluIleGlyGlyValIleSerAlaGlnAsnGlnGln
305310315320
AlaLysGlyGlyLysLeuMetIleThrGlyAspLysValThrLeuLys
325330335
ThrGlyAlaValIleAspLeuSerGlyLysGluGlyGlyGluThrTyr
340345350
LeuGlyGlyAspGluArgGlyGluGlyLysAsnGlyIleGlnLeuAla
355360365
LysLysThrSerLeuGluLysGlySerThrIleAsnValSerGlyLys
370375380
GluLysGlyGlyArgAlaIleValTrpGlyAspIleAlaLeuIleAsp
385390395400
GlyAsnIleAsnAlaGlnGlySerGlyAspIleAlaLysThrGlyGly
405410415
PheValGluThrSerGlyHisAspLeuPheIleLysAspAsnAlaIle
420425430
ValAspAlaLysGluTrpLeuLeuAspPheAspAsnValSerIleAsn
435440445
AlaGluThrAlaGlyArgSerAsnThrSerGluAspAspGluTyrThr
450455460
GlySerGlyAsnSerAlaSerThrProLysArgAsnLysGluLysThr
465470475480
ThrLeuThrAsnThrThrLeuGluSerIleLeuLysLysGlyThrPhe
485490495
ValAsnIleThrAlaAsnGlnArgIleTyrValAsnSerSerIleAsn
500505510
LeuSerAsnGlySerLeuThrLeuTrpSerGluGlyArgSerGlyGly
515520525
GlyValGluIleAsnAsnAspIleThrThrGlyAspAspThrArgGly
530535540
AlaAsnLeuThrIleTyrSerGlyGlyTrpValAspValHisLysAsn
545550555560
IleSerLeuGlyAlaGlnGlyAsnIleAsnIleThrAlaLysGlnAsp
565570575
IleAlaPheGluLysGlySerAsnGlnValIleThrGlyGlnGlyThr
580585590
IleThrSerGlyAsnGlnLysGlyPheArgPheAsnAsnValSerLeu
595600605
AsnGlyThrGlySerGlyLeuGlnPheThrThrLysArgThrAsnLys
610615620
TyrAlaIleThrAsnLysPheGluGlyThrLeuAsnIleSerGlyLys
625630635640
ValAsnIleSerMetValLeuProLysAsnGluSerGlyTyrAspLys
645650655
PheLysGlyArgThrTyrTrpAsnLeuThrSerLeuAsnValSerGlu
660665670
SerGlyGluPheAsnLeuThrIleAspSerArgGlySerAspSerAla
675680685
GlyThrLeuThrGlnProTyrAsnLeuAsnGlyIleSerPheAsnLys
690695700
AspThrThrPheAsnValGluArgAsnAlaArgValAsnPheAspIle
705710715720
LysAlaProIleGlyIleAsnLysTyrSerSerLeuAsnTyrAlaSer
725730735
PheAsnGlyAsnIleSerValSerGlyGlyGlySerValAspPheThr
740745750
LeuLeuAlaSerSerSerAsnValGlnThrProGlyValValIleAsn
755760765
SerLysTyrPheAsnValSerThrGlySerSerLeuArgPheLysThr
770775780
SerGlySerThrLysThrGlyPheSerIleGluLysAspLeuThrLeu
785790795800
AsnAlaThrGlyGlyAsnIleThrLeuLeuGlnValGluGlyThrAsp
805810815
GlyMetIleGlyLysGlyIleValAlaLysLysAsnIleThrPheGlu
820825830
GlyGlyAsnIleThrPheGlySerArgLysAlaValThrGluIleGlu
835840845
GlyAsnValThrIleAsnAsnAsnAlaAsnValThrLeuIleGlySer
850855860
AspPheAspAsnHisGlnLysProLeuThrIleLysLysAspValIle
865870875880
IleAsnSerGlyAsnLeuThrAlaGlyGlyAsnIleValAsnIleAla
885890895
GlyAsnLeuThrValGluSerAsnAlaAsnPheLysAlaIleThrAsn
900905910
PheThrPheAsnValGlyGlyLeuPheAspAsnLysGlyAsnSerAsn
915920925
IleSerIleAlaLysGlyGlyAlaArgPheLysAspIleAspAsnSer
930935940
LysAsnLeuSerIleThrThrAsnSerSerSerThrTyrArgThrIle
945950955960
IleSerGlyAsnIleThrAsnLysAsnGlyAspLeuAsnIleThrAsn
965970975
GluGlySerAspThrGluMetGlnIleGlyGlyAspValSerGlnLys
980985990
GluGlyAsnLeuThrIleSerSerAspLysIleAsnIleThrLysGln
99510001005
IleThrIleLysAlaGlyValAspGlyGluAsnSerAspSerAspAla
101010151020
ThrAsnAsnAlaAsnLeuThrIleLysThrLysGluLeuLysLeuThr
1025103010351040
GlnAspLeuAsnIleSerGlyPheAsnLysAlaGluIleThrAlaLys
104510501055
AspGlySerAspLeuThrIleGlyAsnThrAsnSerAlaAspGlyThr
106010651070
AsnAlaLysLysValThrPheAsnGlnValLysAspSerLysIleSer
107510801085
AlaAspGlyHisLysValThrLeuHisSerLysValGluThrSerGly
109010951100
SerAsnAsnAsnThrGluAspSerSerAspAsnAsnAlaGlyLeuThr
1105111011151120
IleAspAlaLysAsnValThrValAsnAsnAsnIleThrSerHisLys
112511301135
AlaValSerIleSerAlaThrSerGlyGluIleThrThrLysThrGly
114011451150
ThrThrIleAsnAlaThrThrGlyAsnValGluIleThrAlaGlnThr
115511601165
GlySerIleLeuGlyGlyIleGluSerSerSerGlySerValThrLeu
117011751180
ThrAlaThrGluGlyAlaLeuAlaValSerAsnIleSerGlyAsnThr
1185119011951200
ValThrValThrAlaAsnSerGlyAlaLeuThrThrLeuAlaGlySer
120512101215
ThrIleLysGlyThrGluSerValThrThrSerSerGlnSerGlyAsp
122012251230
IleGlyGlyThrIleSerGlyGlyThrValGluValLysAlaThrGlu
123512401245
SerLeuThrThrGlnSerAsnSerLysIleLysAlaThrThrGlyGlu
125012551260
AlaAsnValThrSerAlaThrGlyThrIleGlyGlyThrIleSerGly
1265127012751280
AsnThrValAsnValThrAlaAsnAlaGlyAspLeuThrValGlyAsn
128512901295
GlyAlaGluIleAsnAlaThrGluGlyAlaAlaThrLeuThrThrSer
130013051310
SerGlyLysLeuThrThrGluAlaSerSerHisIleThrSerAlaLys
131513201325
GlyGlnValAsnLeuSerAlaGlnAspGlySerValAlaGlySerIle
133013351340
AsnAlaAlaAsnValThrLeuAsnThrThrGlyThrLeuThrThrVal
1345135013551360
LysGlySerAsnIleAsnAlaThrSerGlyThrLeuValIleAsnAla
136513701375
LysAspAlaGluLeuAsnGlyAlaAlaLeuGlyAsnHisThrValVal
138013851390
AsnAlaThrAsnAlaAsnGlySerGlySerValIleAlaThrThrSer
139514001405
SerArgValAsnIleThrGlyAspLeuIleThrIleAsnGlyLeuAsn
141014151420
IleIleSerLysAsnGlyIleAsnThrValLeuLeuLysGlyValLys
1425143014351440
IleAspValLysTyrIleGlnProGlyIleAlaSerValAspGluVal
144514501455
IleGluAlaLysArgIleLeuGluLysValLysAspLeuSerAspGlu
146014651470
GluArgGluAlaLeuAlaLysLeuGlyValSerAlaValArgPheIle
147514801485
GluProAsnAsnThrIleThrValAspThrGlnAsnGluPheAlaThr
149014951500
ArgProLeuSerArgIleValIleSerGluGlyArgAlaCysPheSer
1505151015151520
AsnSerAspGlyAlaThrValCysValAsnIleAlaAspAsnGlyArg
152515301535
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4937 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TAAATATACAAGATAATAAAAATAAATCAAGATTTTTGTGATGACAAACAACAATTACAA60
CACCTTTTTTGCAGTCTATATGCAAATATTTTAAAAAAATAGTATAAATCCGCCATATAA120
AATGGTATAATCTTTCATCTTTCATCTTTAATCTTTCATCTTTCATCTTTCATCTTTCAT180
CTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTT240
CACATGAAATGATGAACCGAGGGAAGGGAGGGAGGGGCAAGAATGAAGAGGGAGCTGAAC300
GAACGCAAATGATAAAGTAATTTAATTGTTCAACTAACCTTAGGAGAAAATATGAACAAG360
ATATATCGTCTCAAATTCAGCAAACGCCTGAATGCTTTGGTTGCTGTGTCTGAATTGGCA420
CGGGGTTGTGACCATTCCACAGAAAAAGGCTTCCGCTATGTTACTATCTTTAGGTGTAAC480
CACTTAGCGTTAAAGCCACTTTCCGCTATGTTACTATCTTTAGGTGTAACATCTATTCCA540
CAATCTGTTTTAGCAAGCGGCTTACAAGGAATGGATGTAGTACACGGCACAGCCACTATG600
CAAGTAGATGGTAATAAAACCATTATCCGCAACAGTGTTGACGCTATCATTAATTGGAAA660
CAATTTAACATCGACCAAAATGAAATGGTGCAGTTTTTACAAGAAAACAACAACTCCGCC720
GTATTCAACCGTGTTACATCTAACCAAATCTCCCAATTAAAAGGGATTTTAGATTCTAAC780
GGACAAGTCTTTTTAATCAACCCAAATGGTATCACAATAGGTAAAGACGCAATTATTAAC840
ACTAATGGCTTTACGGCTTCTACGCTAGACATTTCTAACGAAAACATCAAGGCGCGTAAT900
TTCACCTTCGAGCAAACCAAAGATAAAGCGCTCGCTGAAATTGTGAATCACGGTTTAATT960
ACTGTCGGTAAAGACGGCAGTGTAAATCTTATTGGTGGCAAAGTGAAAAACGAGGGTGTG1020
ATTAGCGTAAATGGTGGCAGCATTTCTTTACTCGCAGGGCAAAAAATCACCATCAGCGAT1080
ATAATAAACCCAACCATTACTTACAGCATTGCCGCGCCTGAAAATGAAGCGGTCAATCTG1140
GGCGATATTTTTGCCAAAGGCGGTAACATTAATGTCCGTGCTGCCACTATTCGAAACCAA1200
GGTAAACTTTCTGCTGATTCTGTAAGCAAAGATAAAAGCGGCAATATTGTTCTTTCCGCC1260
AAAGAGGGTGAAGCGGAAATTGGCGGTGTAATTTCCGCTCAAAATCAGCAAGCTAAAGGC1320
GGCAAGCTGATGATTACAGGCGATAAAGTCACATTAAAAACAGGTGCAGTTATCGACCTT1380
TCAGGTAAAGAAGGGGGAGAAACTTACCTTGGCGGTGACGAGCGCGGCGAAGGTAAAAAC1440
GGCATTCAATTAGCAAAGAAAACCTCTTTAGAAAAAGGCTCAACCATCAATGTATCAGGC1500
AAAGAAAAAGGCGGACGCGCTATTGTGTGGGGCGATATTGCGTTAATTGACGGCAATATT1560
AACGCTCAAGGTAGTGGTGATATCGCTAAAACCGGTGGTTTTGTGGAGACATCGGGGCAT1620
TATTTATCCATTGACAGCAATGCAATTGTTAAAACAAAAGAGTGGTTGCTAGACCCTGAT1680
GATGTAACAATTGAAGCCGAAGACCCCCTTCGCAATAATACCGGTATAAATGATGAATTC1740
CCAACAGGCACCGGTGAAGCAAGCGACCCTAAAAAAAATAGCGAACTCAAAACAACGCTA1800
ACCAATACAACTATTTCAAATTATCTGAAAAACGCCTGGACAATGAATATAACGGCATCA1860
AGAAAACTTACCGTTAATAGCTCAATCAACATCGGAAGCAACTCCCACTTAATTCTCCAT1920
AGTAAAGGTCAGCGTGGCGGAGGCGTTCAGATTGATGGAGATATTACTTCTAAAGGCGGA1980
AATTTAACCATTTATTCTGGCGGATGGGTTGATGTTCATAAAAATATTACGCTTGATCAG2040
GGTTTTTTAAATATTACCGCCGCTTCCGTAGCTTTTGAAGGTGGAAATAACAAAGCACGC2100
GACGCGGCAAATGCTAAAATTGTCGCCCAGGGCACTGTAACCATTACAGGAGAGGGAAAA2160
GATTTCAGGGCTAACAACGTATCTTTAAACGGAACGGGTAAAGGTCTGAATATCATTTCA2220
TCAGTGAATAATTTAACCCACAATCTTAGTGGCACAATTAACATATCTGGGAATATAACA2280
ATTAACCAAACTACGAGAAAGAACACCTCGTATTGGCAAACCAGCCATGATTCGCACTGG2340
AACGTCAGTGCTCTTAATCTAGAGACAGGCGCAAATTTTACCTTTATTAAATACATTTCA2400
AGCAATAGCAAAGGCTTAACAACACAGTATAGAAGCTCTGCAGGGGTGAATTTTAACGGC2460
GTAAATGGCAACATGTCATTCAATCTCAAAGAAGGAGCGAAAGTTAATTTCAAATTAAAA2520
CCAAACGAGAACATGAACACAAGCAAACCTTTACCAATTCGGTTTTTAGCCAATATCACA2580
GCCACTGGTGGGGGCTCTGTTTTTTTTGATATATATGCCAACCATTCTGGCAGAGGGGCT2640
GAGTTAAAAATGAGTGAAATTAATATCTCTAACGGCGCTAATTTTACCTTAAATTCCCAT2700
GTTCGCGGCGATGACGCTTTTAAAATCAACAAAGACTTAACCATAAATGCAACCAATTCA2760
AATTTCAGCCTCAGACAGACGAAAGATGATTTTTATGACGGGTACGCACGCAATGCCATC2820
AATTCAACCTACAACATATCCATTCTGGGCGGTAATGTCACCCTTGGTGGACAAAACTCA2880
AGCAGCAGCATTACGGGGAATATTACTATCGAGAAAGCAGCAAATGTTACGCTAGAAGCC2940
AATAACGCCCCTAATCAGCAAAACATAAGGGATAGAGTTATAAAACTTGGCAGCTTGCTC3000
GTTAATGGGAGTTTAAGTTTAACTGGCGAAAATGCAGATATTAAAGGCAATCTCACTATT3060
TCAGAAAGCGCCACTTTTAAAGGAAAGACTAGAGATACCCTAAATATCACCGGCAATTTT3120
ACCAATAATGGCACTGCCGAAATTAATATAACACAAGGAGTGGTAAAACTTGGCAATGTT3180
ACCAATGATGGTGATTTAAACATTACCACTCACGCTAAACGCAACCAAAGAAGCATCATC3240
GGCGGAGATATAATCAACAAAAAAGGAAGCTTAAATATTACAGACAGTAATAATGATGCT3300
GAAATCCAAATTGGCGGCAATATCTCGCAAAAAGAAGGCAACCTCACGATTTCTTCCGAT3360
AAAATTAATATCACCAAACAGATAACAATCAAAAAGGGTATTGATGGAGAGGACTCTAGT3420
TCAGATGCGACAAGTAATGCCAACCTAACTATTAAAACCAAAGAATTGAAATTGACAGAA3480
GACCTAAGTATTTCAGGTTTCAATAAAGCAGAGATTACAGCCAAAGATGGTAGAGATTTA3540
ACTATTGGCAACAGTAATGACGGTAACAGCGGTGCCGAAGCCAAAACAGTAACTTTTAAC3600
AATGTTAAAGATTCAAAAATCTCTGCTGACGGTCACAATGTGACACTAAATAGCAAAGTG3660
AAAACATCTAGCAGCAATGGCGGACGTGAAAGCAATAGCGACAACGATACCGGCTTAACT3720
ATTACTGCAAAAAATGTAGAAGTAAACAAAGATATTACTTCTCTCAAAACAGTAAATATC3780
ACCGCGTCGGAAAAGGTTACCACCACAGCAGGCTCGACCATTAACGCAACAAATGGCAAA3840
GCAAGTATTACAACCAAAACAGGTGATATCAGCGGTACGATTTCCGGTAACACGGTAAGT3900
GTTAGCGCGACTGGTGATTTAACCACTAAATCCGGCTCAAAAATTGAAGCGAAATCGGGT3960
GAGGCTAATGTAACAAGTGCAACAGGTACAATTGGCGGTACAATTTCCGGTAATACGGTA4020
AATGTTACGGCAAACGCTGGCGATTTAACAGTTGGGAATGGCGCAGAAATTAATGCGACA4080
GAAGGAGCTGCAACCTTAACCGCAACAGGGAATACCTTGACTACTGAAGCCGGTTCTAGC4140
ATCACTTCAACTAAGGGTCAGGTAGACCTCTTGGCTCAGAATGGTAGCATCGCAGGAAGC4200
ATTAATGCTGCTAATGTGACATTAAATACTACAGGCACCTTAACCACCGTGGCAGGCTCG4260
GATATTAAAGCAACCAGCGGCACCTTGGTTATTAACGCAAAAGATGCTAAGCTAAATGGT4320
GATGCATCAGGTGATAGTACAGAAGTGAATGCAGTCAACGCAAGCGGCTCTGGTAGTGTG4380
ACTGCGGCAACCTCAAGCAGTGTGAATATCACTGGGGATTTAAACACAGTAAATGGGTTA4440
AATATCATTTCGAAAGATGGTAGAAACACTGTGCGCTTAAGAGGCAAGGAAATTGAGGTG4500
AAATATATCCAGCCAGGTGTAGCAAGTGTAGAAGAAGTAATTGAAGCGAAACGCGTCCTT4560
GAAAAAGTAAAAGATTTATCTGATGAAGAAAGAGAAACATTAGCTAAACTTGGTGTAAGT4620
GCTGTACGTTTTGTTGAGCCAAATAATACAATTACAGTCAATACACAAAATGAATTTACA4680
ACCAGACCGTCAAGTCAAGTGATAATTTCTGAAGGTAAGGCGTGTTTCTCAAGTGGTAAT4740
GGCGCACGAGTATGTACCAATGTTGCTGACGATGGACAGCCGTAGTCAGTAATTGACAAG4800
GTAGATTTCATCCTGCAATGAAGTCATTTTATTTTCGTATTATTTACTGTGTGGGTTAAA4860
GTTCAGTACGGGCTTTACCCATCTTGTAAAAAATTACGGAGAATACAATAAAGTATTTTT4920
AACAGGTTATTATTATG4937
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1477 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetAsnLysIleTyrArgLeuLysPheSerLysArgLeuAsnAlaLeu
151015
ValAlaValSerGluLeuAlaArgGlyCysAspHisSerThrGluLys
202530
GlySerGluLysProAlaArgMetLysValArgHisLeuAlaLeuLys
354045
ProLeuSerAlaMetLeuLeuSerLeuGlyValThrSerIleProGln
505560
SerValLeuAlaSerGlyLeuGlnGlyMetAspValValHisGlyThr
65707580
AlaThrMetGlnValAspGlyAsnLysThrIleIleArgAsnSerVal
859095
AspAlaIleIleAsnTrpLysGlnPheAsnIleAspGlnAsnGluMet
100105110
ValGlnPheLeuGlnGluAsnAsnAsnSerAlaValPheAsnArgVal
115120125
ThrSerAsnGlnIleSerGlnLeuLysGlyIleLeuAspSerAsnGly
130135140
GlnValPheLeuIleAsnProAsnGlyIleThrIleGlyLysAspAla
145150155160
IleIleAsnThrAsnGlyPheThrAlaSerThrLeuAspIleSerAsn
165170175
GluAsnIleLysAlaArgAsnPheThrPheGluGlnThrLysAspLys
180185190
AlaLeuAlaGluIleValAsnHisGlyLeuIleThrValGlyLysAsp
195200205
GlySerValAsnLeuIleGlyGlyLysValLysAsnGluGlyValIle
210215220
SerValAsnGlyGlySerIleSerLeuLeuAlaGlyGlnLysIleThr
225230235240
IleSerAspIleIleAsnProThrIleThrTyrSerIleAlaAlaPro
245250255
GluAsnGluAlaValAsnLeuGlyAspIlePheAlaLysGlyGlyAsn
260265270
IleAsnValArgAlaAlaThrIleArgAsnGlnGlyLysLeuSerAla
275280285
AspSerValSerLysAspLysSerGlyAsnIleValLeuSerAlaLys
290295300
GluGlyGluAlaGluIleGlyGlyValIleSerAlaGlnAsnGlnGln
305310315320
AlaLysGlyGlyLysLeuMetIleThrGlyAspLysValThrLeuLys
325330335
ThrGlyAlaValIleAspLeuSerGlyLysGluGlyGlyGluThrTyr
340345350
LeuGlyGlyAspGluArgGlyGluGlyLysAsnGlyIleGlnLeuAla
355360365
LysLysThrSerLeuGluLysGlySerThrIleAsnValSerGlyLys
370375380
GluLysGlyGlyPheAlaIleValTrpGlyAspIleAlaLeuIleAsp
385390395400
GlyAsnIleAsnAlaGlnGlySerGlyAspIleAlaLysThrGlyGly
405410415
PheValGluThrSerGlyHisAspLeuPheIleLysAspAsnAlaIle
420425430
ValAspAlaLysGluTrpLeuLeuAspPheAspAsnValSerIleAsn
435440445
AlaGluAspProLeuPheAsnAsnThrGlyIleAsnAspGluPhePro
450455460
ThrGlyThrGlyGluAlaSerAspProLysLysAsnSerGluLeuLys
465470475480
ThrThrLeuThrAsnThrThrIleSerAsnTyrLeuLysAsnAlaTrp
485490495
ThrMetAsnIleThrAlaSerArgLysLeuThrValAsnSerSerIle
500505510
AsnIleGlySerAsnSerHisLeuIleLeuHisSerLysGlyGlnArg
515520525
GlyGlyGlyValGlnIleAspGlyAspIleThrSerLysGlyGlyAsn
530535540
LeuThrIleTyrSerGlyGlyTrpValAspValHisLysAsnIleThr
545550555560
LeuAspGlnGlyPheLeuAsnIleThrAlaAlaSerValAlaPheGlu
565570575
GlyGlyAsnAsnLysAlaArgAspAlaAlaAsnAlaLysIleValAla
580585590
GlnGlyThrValThrIleThrGlyGluGlyLysAspPheArgAlaAsn
595600605
AsnValSerLeuAsnGlyThrGlyLysGlyLeuAsnIleIleSerSer
610615620
ValAsnAsnLeuThrHisAsnLeuSerGlyThrIleAsnIleSerGly
625630635640
AsnIleThrIleAsnGlnThrThrArgLysAsnThrSerTyrTrpGln
645650655
ThrSerHisAspSerHisTrpAsnValSerAlaLeuAsnLeuGluThr
660665670
GlyAlaAsnPheThrPheIleLysTyrIleSerSerAsnSerLysGly
675680685
LeuThrThrGlnTyrArgSerSerAlaGlyValAsnPheAsnGlyVal
690695700
AsnGlyAsnMetSerPheAsnLeuLysGluGlyAlaLysValAsnPhe
705710715720
LysLeuLysProAsnGluAsnMetAsnThrSerLysProLeuProIle
725730735
ArgPheLeuAlaAsnIleThrAlaThrGlyGlyGlySerValPhePhe
740745750
AspIleTyrAlaAsnHisSerGlyArgGlyAlaGluLeuLysMetSer
755760765
GluIleAsnIleSerAsnGlyAlaAsnPheThrLeuAsnSerHisVal
770775780
ArgGlyAspAspAlaPheLysIleAsnLysAspLeuThrIleAsnAla
785790795800
ThrAsnSerAsnPheSerLeuArgGlnThrLysAspAspPheTyrAsp
805810815
GlyTyrAlaArgAsnAlaIleAsnSerThrTyrAsnIleSerIleLeu
820825830
GlyGlyAsnValThrLeuGlyGlyGlnAsnSerSerSerSerIleThr
835840845
GlyAsnIleThrIleGluLysAlaAlaAsnValThrLeuGluAlaAsn
850855860
AsnAlaProAsnGlnGlnAsnIleArgAspArgValIleLysLeuGly
865870875880
SerLeuLeuValAsnGlySerLeuSerLeuThrGlyGluAsnAlaAsp
885890895
IleLysGlyAsnLeuThrIleSerGluSerAlaThrPheLysGlyLys
900905910
ThrArgAspThrLeuAsnIleThrGlyAsnPheThrAsnAsnGlyThr
915920925
AlaGluIleAsnIleThrGlnGlyValValLysLeuGlyAsnValThr
930935940
AsnAspGlyAspLeuAsnIleThrThrHisAlaLysArgAsnGlnArg
945950955960
SerIleIleGlyGlyAspIleIleAsnLysLysGlySerLeuAsnIle
965970975
ThrAspSerAsnAsnAspAlaGluIleGlnIleGlyGlyAsnIleSer
980985990
GlnLysGluGlyAsnLeuThrIleSerSerAspLysIleAsnIleThr
99510001005
LysGlnIleThrIleLysLysGlyIleAspGlyGluAspSerSerSer
101010151020
AspAlaThrSerAsnAlaAsnLeuThrIleLysThrLysGluLeuLys
1025103010351040
LeuThrGluAspLeuSerIleSerGlyPheAsnLysAlaGluIleThr
104510501055
AlaLysAspGlyArgAspLeuThrIleGlyAsnSerAsnAspGlyAsn
106010651070
SerGlyAlaGluAlaLysThrValThrPheAsnAsnValLysAspSer
107510801085
LysIleSerAlaAspGlyHisAsnValThrLeuAsnSerLysValLys
109010951100
ThrSerSerSerAsnGlyGlyArgGluSerAsnSerAspAsnAspThr
1105111011151120
GlyLeuThrIleThrAlaLysAsnValGluValAsnLysAspIleThr
112511301135
SerLeuLysThrValAsnIleThrAlaSerGluLysValThrThrThr
114011451150
AlaGlySerThrIleAsnAlaThrAsnGlyLysAlaSerIleThrThr
115511601165
LysThrGlyAspIleSerGlyThrIleSerGlyAsnThrValSerVal
117011751180
SerAlaThrValAspLeuThrThrLysSerGlySerLysIleGluAla
1185119011951200
LysSerGlyGluAlaAsnValThrSerAlaThrGlyThrIleGlyGly
120512101215
ThrIleSerGlyAsnThrValAsnValThrAlaAsnAlaGlyAspLeu
122012251230
ThrValGlyAsnGlyAlaGluIleAsnAlaThrGluGlyAlaAlaThr
123512401245
LeuThrAlaThrGlyAsnThrLeuThrThrGluAlaGlySerSerIle
125012551260
ThrSerThrLysGlyGlnValAspLeuLeuAlaGlnAsnGlySerIle
1265127012751280
AlaGlySerIleAsnAlaAlaAsnValThrLeuAsnThrThrGlyThr
128512901295
LeuThrThrValAlaGlySerAspIleLysAlaThrSerGlyThrLeu
130013051310
ValIleAsnAlaLysAspAlaLysLeuAsnGlyAspAlaSerGlyAsp
131513201325
SerThrGluValAsnAlaValAsnAlaSerGlySerGlySerValThr
133013351340
AlaAlaThrSerSerSerValAsnIleThrGlyAspLeuAsnThrVal
1345135013551360
AsnGlyLeuAsnIleIleSerLysAspGlyArgAsnThrValArgLeu
136513701375
ArgGlyLysGluIleGluValLysTyrIleGlnProGlyValAlaSer
138013851390
ValGluGluValIleGluAlaLysArgValLeuGluLysValLysAsp
139514001405
LeuSerAspGluGluArgGluThrLeuAlaLysLeuGlyValSerAla
141014151420
ValArgPheValGluProAsnAsnThrIleThrValAsnThrGlnAsn
1425143014351440
GluPheThrThrArgProSerSerGlnValIleIleSerGluGlyLys
144514501455
AlaCysPheSerSerGlyAsnGlyAlaArgValCysThrAsnValAla
146014651470
AspAspGlyGlnPro
1475
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9171 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ACAGCGTTCTCTTAATACTAGTACAAACCCACAATAAAATATGACAAACAACAATTACAA60
CACCTTTTTTGCAGTCTATATGCAAATATTTTAAAAAATAGTATAAATCCGCCATATAAA120
ATGGTATAATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATC180
TTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTC240
ACATGAAATGATGAACCGAGGGAAGGGAGGGAGGGGCAAGAATGAAGAGGGAGCTGAACG300
AACGCAAATGATAAAGTAATTTAATTGTTCAACTAACCTTAGGAGAAAATATGAACAAGA360
TATATCGTCTCAAATTCAGCAAACGCCTGAATGCTTTGGTTGCTGTGTCTGAATTGGCAC420
GGGGTTGTGACCATTCCACAGAAAAAGGCAGCGAAAAACCTGCTCGCATGAAAGTGCGTC480
ACTTAGCGTTAAAGCCACTTTCCGCTATGTTACTATCTTTAGGTGTAACATCTATTCCAC540
AATCTGTTTTAGCAAGCGGCTTACAAGGAATGGATGTAGTACACGGCACAGCCACTATGC600
AAGTAGATGGTAATAAAACCATTATCCGCAACAGTGTTGACGCTATCATTAATTGGAAAC660
AATTTAACATCGACCAAAATGAAATGGTGCAGTTTTTACAAGAAAACAACAACTCCGCCG720
TATTCAACCGTGTTACATCTAACCAAATCTCCCAATTAAAAGGGATTTTAGATTCTAACG780
GACAAGTCTTTTTAATCAACCCAAATGGTATCACAATAGGTAAAGACGCAATTATTAACA840
CTAATGGCTTTACGGCTTCTACGCTAGACATTTCTAACGAAAACATCAAGGCGCGTAATT900
TCACCTTCGAGCAAACCAAAGATAAAGCGCTCGCTGAAATTGTGAATCACGGTTTAATTA960
CTGTCGGTAAAGACGGCAGTGTAAATCTTATTGGTGGCAAAGTGAAAAACGAGGGTGTGA1020
TTAGCGTAAATGGTGGCAGCATTTCTTTACTCGCAGGGCAAAAAATCACCATCAGCGATA1080
TAATAAACCCAACCATTACTTACAGCATTGCCGCGCCTGAAAATGAAGCGGTCAATCTGG1140
GCGATATTTTTGCCAAAGGCGGTAACATTAATGTCCGTGCTGCCACTATTCGAAACCAAG1200
CTTTCCGCCAAAGAGGGTGAAGCGGAAATTGGCGGTGTAATTTCCGCTCAAAATCAGCAA1260
GCTAAAGGCGGCAAGCTGATGATTACAGGCGATAAAGTCACATTAAAAACAGGTGCAGTT1320
ATCGACCTTTCAGGTAAAGAAGGGGGAGAAACTTACCTTGGCGGTGACGAGCGCGGCGAA1380
GGTAAAAACGGCATTCAATTAGCAAAGAAAACCTCTTTAGAAAAAGGCTCAACCATCAAT1440
GTATCAGGCAAAGAAAAAGGCGGACGCGCTATTGTGTGGGGCGATATTGCGTTAATTGAC1500
GGCAATATTAACGCTCAAGGTAGTGGTGATATCGCTAAAACCGGTGGTTTTGTGGAGACG1560
TCGGGGCATGATTTATTCATCAAAGACAATGCAATTGTTGACGCCAAAGAGTGGTTGTTA1620
GACCCGGATAATGTATCTATTAATGCAGAAACAGCAGGACGCAGCAATACTTCAGAAGAC1680
GATGAATACACGGGATCCGGGAATAGTGCCAGCACCCCAAAACGAAACAAAGAAAAGACA1740
ACATTAACAAACACAACTCTTGAGAGTATACTAAAAAAAGGTACCTTTGTTAACATCACT1800
GCTAATCAACGCATCTATGTCAATAGCTCCATTAATTTATCCAATGGCAGCTTAACTCTT1860
TGGAGTGAGGGTCGGAGCGGTGGCGGCGTTGAGATTAACAACGATATTACCACCGGTGAT1920
GATACCAGAGGTGCAAACTTAACAATTTACTCAGGCGGCTGGGTTGATGTTCATAAAAAT1980
ATCTCACTCGGGGCGCAAGGTAACATAAACATTACAGCTAAACAAGATATCGCCTTTGAG2040
AAAGGAAGCAACCAAGTCATTACAGGTCAAGGGACTATTACCTCAGGCAATCAAAAAGGT2100
TTTAGATTTAATAATGTCTCTCTAAACGGCACTGGCAGCGGACTGCAATTCACCACTAAA2160
AGAACCAATAAATACGCTATCACAAATAAATTTGAAGGGACTTTAAATATTTCAGGGAAA2220
GTGAACATCTCAATGGTTTTACCTAAAAATGAAAGTGGATATGATAAATTCAAAGGACGC2280
ACTTACTGGAATTTAACCTCGAAAGTGGATATGATAAATTCAAAGGACGCCCTCACTATT2340
GACTCCAGAGGAAGCGATAGTGCAGGCACACTTACCCAGCCTTATAATTTAAACGGTATA2400
TCATTCAACAAAGACACTACCTTTAATGTTGAACGAAATGCAAGAGTCAACTTTGACATC2460
AAGGCACCAATAGGGATAAATAAGTATTCTAGTTTGAATTACGCATCATTTAATGGAAAC2520
ATTTCAGTTTCGGGAGGGGGGAGTGTTGATTTCACACTTCTCGCCTCATCCTCTAACGTC2580
CAAACCCCCGGTGTAGTTATAAATTCTAAATACTTTAATGTTTCAACAGGGTCAAGTTTA2640
AGATTTAAAACTTCAGGCTCAACAAAAACTGGCTTCTCAATAGAGAAAGATTTAACTTTA2700
AATGCCACCGGAGGCAACATAACACTTTTGCAAGTTGAAGGCACCGATGGAATGATTGGT2760
AAAGGCATTGTAGCCAAAAAAAACATAACCTTTGAAGGAGGTAAGATGAGGTTTGGCTCC2820
AGGAAAGCCGTAACAGAAATCGAAGGCAATGTTACTATCAATAACAACGCTAACGTCACT2880
CTTATCGGTTCGGATTTTGACAACCATCAAAAACCTTTAACTATTAAAAAAGATGTCATC2940
ATTAATAGCGGCAACCTTACCGCTGGAGGCAATATTGTCAATATAGCCGGAAATCTTACC3000
GTTGAAAGTAACGCTAATTTCAAAGCTATCACAAATTTCACTTTTAATGTAGGCGGCTTG3060
TTTGACAACAAAGGCAATTCAAATATTTCCATTGCCAAAGGAGGGGCTCGCTTTAAAGAC3120
ATTGATAATTCCAAGAATTTAAGCATCACCACCAACTCCAGCTCCACTTACCGCACTATT3180
ATAAGCGGCAATATAACCAATAAAAACGGTGATTTAAATATTACGAACGAAGGTAGTGAT3240
ACTGAAATGCAAATTGGCGGCGATGTCTCGCAAAAAGAAGGTAATCTCACGATTTCTTCT3300
GACAAAATCAATATTACCAAACAGATAACAATCAAGGCAGGTGTTGATGGGGAGAATTCC3360
GATTCAGACGCGACAAACAATGCCAATCTAACCATTAAAACCAAAGAATTGAAATTAACG3420
CAAGACCTAAATATTTCAGGTTTCAATAAAGCAGAGATTACAGCTAAAGATGGTAGTGAT3480
TTAACTATTGGTAACACCAATAGTGCTGATGGTACTAATGCCAAAAAAGTAACCTTTAAC3540
CAGGTTAAAGATTCAAAAATCTCTGCTGACGGTCACAAGGTGACACTACACAGCAAAGTG3600
GAAACATCCGGTAGTAATAACAACACTGAAGATAGCAGTGACAATAATGCCGGCTTAACT3660
ATCGATGCAAAAAATGTAACAGTAAACAACAATATTACTTCTCACAAAGCAGTGAGCATC3720
TCTGCGACAAGTGGAGAAATTACCACTAAAACAGGTACAACCATTAACGCAACCACTGGT3780
AACGTGGAGATAACCGCTCAAACAGGTAGTATCCTAGGTGGAATTGAGTCCAGCTCTGGC3840
TCTGTAACACTTACTGCAACCGAGGGCGCTCTTGCTGTAAGCAATATTTCGGGCAACACC3900
GTTACTGTTACTGCAAATAGCGGTGCATTAACCACTTTGGCAGGCTCTACAATTAAAGGA3960
ACCGAGAGTGTAACCACTTCAAGTCAATCAGGCGATATCGGCGGTACGATTTCTGGTGGC4020
ACAGTAGAGGTTAAAGCAACCGAAAGTTTAACCACTCAATCCAATTCAAAAATTAAAGCA4080
ACAACAGGCGAGGCTAACGTAACAAGTGCAACAGGTACAATTGGTGGTACGATTTCCGGT4140
AATACGGTAAATGTTACGGCAAACGCTGGCGATTTAACAGTTGGGAATGGCGCAGAAATT4200
AATGCGACAGAAGGAGCTGCAACCTTAACTACATCATCGGGCAAATTAACTACCGAAGCT4260
AGTTCACACATTACTTCAGCCAAGGGTCAGGTAAATCTTTCAGCTCAGGATGGTAGCGTT4320
GCAGGAAGTATTAATGCCGCCAATGTGACACTAAATACTACAGGCACTTTAACTACCGTG4380
AAGGGTTCAAACATTAATGCAACCAGCGGTACCTTGGTTATTAACGCAAAAGACGCTGAG4440
CTAAATGGCGCAGCATTGGGTAACCACACAGTGGTAAATGCAACCAACGCAAATGGCTCC4500
GGCAGCGTAATCGCGACAACCTCAAGCAGAGTGAACATCACTGGGGATTTAATCACAATA4560
AATGGATTAAATATCATTTCAAAAAACGGTATAAACACCGTACTGTTAAAAGGCGTTAAA4620
ATTGATGTGAAATACATTCAACCGGGTATAGCAAGCGTAGATGAAGTAATTGAAGCGAAA4680
CGCATCCTTGAGAAGGTAAAAGATTTATCTGATGAAGAAAGAGAAGCGTTAGCTAAACTT4740
GGCGTAAGTGCTGTACGTTTTATTGAGCCAAATAATACAATTACAGTCGATACACAAAAT4800
GAATTTGCAACCAGACCATTAAGTCGAATAGTGATTTCTGAAGGCAGGGCGTGTTTCTCA4860
AACAGTGATGGCGCGACGGTGTGCGTTAATATCGCTGATAACGGGCGGTAGCGGTCAGTA4920
ATTGACAAGGTAGATTTCATCCTGCAATGAAGTCATTTTATTTTCGTATTATTTACTGTG4980
TGGGTTAAAGTTCAGTACGGGCTTTACCCATCTTGTAAAAAATTACGGAGAATACAATAA5040
AGTATTTTTAACAGGTTATTATTATGAAAAATATAAAAAGCAGATTAAAACTCAGTGCAA5100
TATCAGTATTGCTTGGCCTGGCTTCTTCATCATTGTATGCAGAAGAAGCGTTTTTAGTAA5160
AAGGCTTTCAGTTATCTGGTGCACTTGAAACTTTAAGTGAAGACGCCCAACTGTCTGTAG5220
CAAAATCTTTATCTAAATACCAAGGCTCGCAAACTTTAACAAACCTAAAAACAGCACAGC5280
TTGAATTACAGGCTGTGCTAGATAAGATTGAGCCAAATAAGTTTGATGTGATATTGCCAC5340
AACAAACCATTACGGATGGCAATATTATGTTTGAGCTAGTCTCGAAATCAGCCGCAGAAA5400
GCCAAGTTTTTTATAAGGCGAGCCAGGGTTATAGTGAAGAAAATATCGCTCGTAGCCTGC5460
CATCTTTGAAACAAGGAAAAGTGTATGAAGATGGTCGTCAGTGGTTCGATTTGCGTGAAT5520
TCAATATGGCAAAAGAAAATCCACTTAAAGTCACTCGCGTGCATTACGAGTTAAACCCTA5580
AAAACAAAACCTCTGATTTGGTAGTTGCAGGTTTTTCGCCTTTTGGCAAAACGCGTAGCT5640
TTGTTTCCTATGATAATTTCGGCGCAAGGGAGTTTAACTATCAACGTGTAAGTCTAGGTT5700
TTGTAAATGCCAATTTGACCGGACATGATGATGTATTAAATCTAAACGCATTGACCAATG5760
TAAAAGCACCATCAAAATCTTATGCGGTAGGCATAGGATATACTTATCCGTTTTATGATA5820
AACACCAATCCTTAAGTCTTTATACCAGCATGAGTTATGCTGATTCTAATGATATCGACG5880
GCTTACCAAGTGCGATTAATCGTAAATTATCAAAAGGTCAATCTATCTCTGCGAATCTGA5940
AATGGAGTTATTATCTCCCGACATTTAACCTTGGAATGGAAGACCAGTTTAAAATTAATT6000
TAGGCTACAACTACCGCCATATTAATCAAACATCCGAGTTAAACACCCTGGGTGCAACGA6060
AGAAAAAATTTGCAGTATCAGGCGTAAGTGCAGGCATTGATGGACATATCCAATTTACCC6120
CTAAAACAATCTTTAATATTGATTTAACTCATCATTATTACGCGAGTAAATTACCAGGCT6180
CTTTTGGAATGGAGCGCATTGGCGAAACATTTAATCGCAGCTATCACATTAGCACAGCCA6240
GTTTAGGGTTGAGTCAAGAGTTTGCTCAAGGTTGGCATTTTAGCAGTCAATTATCGGGTC6300
AGTTTACTCTACAAGATATAAGTAGCATAGATTTATTCTCTGTAACAGGTACTTATGGCG6360
TCAGAGGCTTTAAATACGGCGGTGCAAGTGGTGAGCGCGGTCTTGTATGGCGTAATGAAT6420
TAAGTATGCCAAAATACACCCGCTTTCAAATCAGCCCTTATGCGTTTTATGATGCAGGTC6480
AGTTCCGTTATAATAGCGAAAATGCTAAAACTTACGGCGAAGATATGCACACGGTATCCT6540
CTGCGGGTTTAGGCATTAAAACCTCTCCTACACAAAACTTAAGCTTAGATGCTTTTGTTG6600
CTCGTCGCTTTGCAAATGCCAATAGTGACAATTTGAATGGCAACAAAAAACGCACAAGCT6660
CACCTACAACCTTCTGGGGTAGATTAACATTCAGTTTCTAACCCTGAAATTTAATCAACT6720
GGTAAGCGTTCCGCCTACCAGTTTATAACTATATGCTTTACCCGCCAATTTACAGTCTAT6780
ACGCAACCCTGTTTTCATCCTTATATATCAAACAAACTAAGCAAACCAAGCAAACCAAGC6840
AAACCAAGCAAACCAAGCAAACCAAGCAAACCAAGCAAACCAAGCAAACCAAGCAAACCA6900
AGCAAACCAAGCAAACCAAGCAAACCAAGCAAACCAAGCAATGCTAAAAAACAATTTATA6960
TGATAAACTAAAACATACTCCATACCATGGCAATACAAGGGATTTAATAATATGACAAAA7020
GAAAATTTACAAAGTGTTCCACAAAATACGACCGCTTCACTTGTAGAATCAAACAACGAC7080
CAAACTTCCCTGCAAATACTTAAACAACCACCCAAACCCAACCTATTACGCCTGGAACAA7140
CATGTCGCCAAAAAAGATTATGAGCTTGCTTGCCGCGAATTAATGGCGATTTTGGAAAAA7200
ATGGACGCTAATTTTGGAGGCGTTCACGATATTGAATTTGACGCACCTGCTCAGCTGGCA7260
TATCTACCCGAAAAACTACTAATTCATTTTGCCACTCGTCTCGCTAATGCAATTACAACA7320
CTCTTTTCCGACCCCGAATTGGCAATTTCCGAAGAAGGGGCATTAAAGATGATTAGCCTG7380
CAACGCTGGTTGACGCTGATTTTTGCCTCTTCCCCCTACGTTAACGCAGACCATATTCTC7440
AATAAATATAATATCAACCCAGATTCCGAAGGTGGCTTTCATTTAGCAACAGACAACTCT7500
TCTATTGCTAAATTCTGTATTTTTTACTTACCCGAATCCAATGTCAATATGAGTTTAGAT7560
GCGTTATGGGCAGGGAATCAACAACTTTGTGCTTCATTGTGTTTTGCGTTGCAGTCTTCA7620
CGTTTTATTGGTACTGCATCTGCGTTTCATAAAAGAGCGGTGGTTTTACAGTGGTTTCCT7680
AAAAAACTCGCCGAAATTGCTAATTTAGATGAATTGCCTGCAAATATCCTTCATGATGTA7740
TATATGCACTGCAGTTATGATTTAGCAAAAAACAAGCACGATGTTAAGCGTCCATTAAAC7800
GAACTTGTCCGCAAGCATATCCTCACGCAAGGATGGCAAGACCGCTACCTTTACACCTTA7860
GGTAAAAAGGACGGCAAACCTGTGATGATGGTACTGCTTGAACATTTTAATTCGGGACAT7920
TCGATTTATCGCACGCATTCAACTTCAATGATTGCTGCTCGAGAAAAATTCTATTTAGTC7980
GGCTTAGGCCATGAGGGCGTTGATAACATAGGTCGAGAAGTGTTTGACGAGTTCTTTGAA8040
ATCAGTAGCAATAATATAATGGAGAGACTGTTTTTTATCCGTAAACAGTGCGAAACTTTC8100
CAACCCGCAGTGTTCTATATGCCAAGCATTGGCATGGATATTACCACGATTTTTGTGAGC8160
AACACTCGGCTTGCCCCTATTCAAGCTGTAGCCTTGGGTCATCCTGCCACTACGCATTCT8220
GAATTTATTGATTATGTCATCGTAGAAGATGATTATGTGGGCAGTGAAGATTGTTTTAGC8280
GAAACCCTTTTACGCTTACCCAAAGATGCCCTACCTTATGTACCATCTGCACTCGCCCCA8340
CAAAAAGTGGATTATGTACTCAGGGAAAACCCTGAAGTAGTCAATATCGGTATTGCCGCT8400
ACCACAATGAAATTAAACCCTGAATTTTTGCTAACATTGCAAGAAATCAGAGATAAAGCT8460
AAAGTCAAAATACATTTTCATTTCGCACTTGGACAATCAACAGGCTTGACACACCCTTAT8520
GTCAAATGGTTTATCGAAAGCTATTTAGGTGACGATGCCACTGCACATCCCCACGCACCT8580
TATCACGATTATCTGGCAATATTGCGTGATTGCGATATGCTACTAAATCCGTTTCCTTTC8640
GGTAATACTAACGGCATAATTGATATGGTTACATTAGGTTTAGTTGGTGTATGCAAAACG8700
GGGGATGAAGTACATGAACATATTGATGAAGGTCTGTTTAAACGCTTAGGACTACCAGAA8760
TGGCTGATAGCCGACACACGAGAAACATATATTGAATGTGCTTTGCGTCTAGCAGAAAAC8820
CATCAAGAACGCCTTGAACTCCGTCGTTACATCATAGAAAACAACGGCTTACAAAAGCTT8880
TTTACAGGCGACCCTCGTCCATTGGGCAAAATACTGCTTAAGAAAACAAATGAATGGAAG8940
CGGAAGCACTTGAGTAAAAAATAACGGTTTTTTAAAGTAAAAGTGCGGTTAATTTTCAAA9000
GCGTTTTAAAAACCTCTCAAAAATCAACCGCACTTTTATCTTTATAACGCTCCCGCGCGC9060
TGACAGTTTATCTCTTTCTTAAAATACCCATAAAATTGTGGCAATAGTTGGGTAATCAAA9120
TTCAATTGTTGATACGGCAAACTAAAGACGGCGCGTTCTTCGGCAGTCATC9171
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9323 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGCCACTTCAATTTTGGATTGTTGAAATTCAACTAACCAAAAAGTGCGGTTAAAATCTGT60
GGAGAAAATAGGTTGTAGTGAAGAACGAGGTAATTGTTCAAAAGGATAAAGCTCTCTTAA120
TTGGGCATTGGTTGGCGTTTCTTTTTCGGTTAATAGTAAATTATATTCTGGACGACTATG180
CAATCCACCAACAACTTTACCGTTGGTTTTAAGCGTTAATGTAAGTTCTTGCTCTTCTTG240
GCGAATACGTAATCCCATTTTTTGTTTAGCAAGAAAATGATCGGGATAATCATAATAGGT300
GTTGCCCAAAAATAAATTTTGATGTTCTAAAATCATAAATTTTGCAAGATATTGTGGCAA360
TTCAATACCTATTTGTGGCGAAATCGCCAATTTTAATTCAATTTCTTGTAGCATAATATT420
TCCCACTCAAATCAACTGGTTAAATATACAAGATAATAAAAATAAATCAAGATTTTTGTG480
ATGACAAACAACAATTACAACACCTTTTTTGCAGTCTATATGCAAATATTTTAAAAAAAT540
AGTATAAATCCGCCATATAAAATGGTATAATCTTTCATCTTTCATCTTTCATCTTTCATC600
TTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTCATCTTTC660
ATCTTTCATCTTTCATCTTTCACATGAAATGATGAACCGAGGGAAGGGAGGGAGGGGCAA720
GAATGAAGAGGGAGCTGAACGAACGCAAATGATAAAGTAATTTAATTGTTCAACTAACCT780
TAGGAGAAAATATGAACAAGATATATCGTCTCAAATTCAGCAAACGCCTGAATGCTTTGG840
TTGCTGTGTCTGAATTGGCACGGGGTTGTGACCATTCCACAGAAAAAGGCAGCGAAAAAC900
CTGCTCGCATGAAAGTGCGTCACTTAGCGTTAAAGCCACTTTCCGCTATGTTACTATCTT960
TAGGTGTAACATCTATTCCACAATCTGTTTTAGCAAGCGGCAATTTAACATCGACCAAAA1020
TGAAATGGTGCAGTTTTTACAAGAAAACAAGTAATAAAACCATTATCCGCAACAGTGTTG1080
ACGCTATCATTAATTGGAAACAATTTAACATCGACCAAAATGAAATGGTGCAGTTTTTAC1140
AAGAAAACAACAACTCCGCCGTATTCAACCGTGTTACATCTAACCAAATCTCCCAATTAA1200
AAGGGATTTTAGATTCTAACGGACAAGTCTTTTTAATCAACCCAAATGGTATCACAATAG1260
GTAAAGACGCAATTATTAACACTAATGGCTTTACGGCTTCTACGCTAGACATTTCTAACG1320
AAAACATCAAGGCGCGTAATTTCACCTTCGAGCAAACCAAAGATAAAGCGCTCGCTGAAA1380
TTGTGAATCACGGTTTAATTACTGTCGGTAAAGACGGCAGTGTAAATCTTATTGGTGGCA1440
AAGTGAAAAACGAGGGTGTGATTAGCGTAAATGGTGGCAGCATTTCTTTACTCGCAGGGC1500
AAAAAATCACCATCAGCGATATAATAAACCCAACCATTACTTACAGCATTGCCGCGCCTG1560
AAAATGAAGCGGTCAATCTGGGCGATATTTTTGCCAAAGGCGGTAACATTAATGTCCGTG1620
CTGCCACTATTCGAAACCAAGGTAAACTTTCTGCTGATTCTGTAAGCAAAGATAAAAGCG1680
GCAATATTGTTCTTTCCGCCAAAGAGGGTGAAGCGGAAATTGGCGGTGTAATTTCCGCTC1740
AAAATCAGCAAGCTAAAGGCGGCAAGCTGATGATAAAGTCCGATAAAGTCACATTAAAAA1800
CAGGTGCAGTTATCGACCTTTCAGGTAAAGAAGGGGGAGAAACTTACCTTGGCGGTGACG1860
AGCGCGGCGAAGGTAAAAACGGCATTCAATTAGCAAAGAAAACCTCTTTAGAAAAAGGCT1920
CAACCATCAATGTATCAGGCAAAGAAAAAGGCGGACGCGCTATTGTGTGGGGCGATATTG1980
CGTTAATTGACGGCAATATTAACGCTCAAGGTAGTGGTGATATCGCTAAAACCGGTGGTT2040
TTGTGGAGACATCGGGGCATTATTTATCCATTGACAGCAATGCAATTGTTAAAACAAAAG2100
AGTGGTTGCTAGACCCTGATGATGTAACAATTGAAGCCGAAGACCCCCTTCGCAATAATA2160
CCGGTATAAATGATGAATTCCCAACAGGCACCGGTGAAGCAAGCGACCCTAAAAAAAATA2220
GCGAACTCAAAACAACGCTAACCAATACAACTATTTCAAATTATCTGAAAAACGCCTGGA2280
CAATGAATATAACGGCATCAAGAAAACTTACCGTTAATAGCTCAATCAACATCGGAAGCA2340
ACTCCCACTTAATTCTCCATAGTAAAGGTCAGCGTGGCGGAGGCGTTCAGATTGATGGAG2400
ATATTACTTCTAAAGGCGGAAATTTAACCATTTATTCTGGCGGATGGGTTGATGTTCATA2460
AAAATATTACGCTTGATCAGGGTTTTTTAAATATTACCGCCGCTTCCGTAGCTTTTGAAG2520
GTGGAAATAACAAAGCACGCGACGCGGCAAATGCTAAAATTGTCGCCCAGGGCACTGTAA2580
CCATTACAGGAGAGGGAAAAGATTTCAGGGCTAACAACGTATCTTTAAACGGAACGGGTA2640
AAGGTCTGAATATCATTTCATCAGTGAATAATTTAACCCACAATCTTAGTGGCACAATTA2700
ACATATCTGGGAATATAACAATTAACCAAACTACGAGAAAGAACACCTCGTATTGGCAAA2760
CCAGCCATGATTCGCACTGGAACGTCAGTGCTCTTAATCTAGAGACAGGCGCAAATTTTA2820
CCTTTATTAAATACATTTCAAGCAATAGCAAAGGCTTAACAACACAGTATAGAAGCTCTG2880
CAGGGGTGAATTTTAACGGCGTAAATGGCAACATGTCATTCAATCTCAAAGAAGGAGCGA2940
AAGTTAATTTCAAATTAAAACCAAACGAGAACATGAACACAAGCAAACCTTTACCAATTC3000
GGTTTTTAGCCAATATCACAGCCACTGGTGGGGGCTCTGTTTTTTTTGATATATATGCCA3060
ACCATTCTGGCAGAGGGGCTGAGTTAAAAATGAGTGAAATTAATATCTCTAACGGCGCTA3120
ATTTTACCTTAAATTCCCATGTTCGCGGCGATGACGCTTTTAAAATCAACAAAGACTTAA3180
CCATAAATGCAACCAATTCAAATTTCAGCCTCAGACAGACGAAAGATGATTTTTATGACG3240
GGTACGCACGCAATGCCATCAATTCAACCTACAACATATCCATTCTGGGCGGTAATGTCA3300
CCCTTGGTGGACAAAACTCAAGCAGCAGCATTACGGGGAATATTACTATCGAGAAAGCAG3360
CAAATGTTACGCTAGAAGCCAATAACGCCCCTAATCAGCAAAACATAAGGGATAGAGTTA3420
TAAAACTTGGCAGCTTGCTCGTTAATGGGAGTTTAAGTTTAACTGGCGAAAATGCAGATA3480
TTAAAGGCAATCTCACTATTTCAGAAAGCGCCACTTTTAAAGGAAAGACTAGAGATACCC3540
TAAATATCACCGGCAATTTTACCAATAATGGCACTGCCGAAATTAATATAACACAAGGAG3600
TGGTAAAACTTGGCAATGTTACCAATGATGGTGATTTAAACATTACCACTCACGCTAAAC3660
GCAACCAAAGAAGCATCATCGGCGGAGATATAATCAACAAAAAAGGAAGCTTAAATATTA3720
CAGACAGTAATAATGATGCTGAAATCCAAATTGGCGGCAATATCTCGCAAAAAGAAGGCA3780
ACCTCACGATTTCTTCCGATAAAATTAATATCACCAAACAGATAACAATCAAAAAGGGTA3840
TTGATGGAGAGGACTCTAGTTCAGATGCGACAAGTAATGCCAACCTAACTATTAAAACCA3900
AAGAATTGAAATTGACAGAAGACCTAAGTATTTCAGGTTTCAATAAAGCAGAGATTACAG3960
CCAAAGATGGTAGAGATTTAACTATTGGCAACAGTAATGACGGTAACAGCGGTGCCGAAG4020
CCAAAACAGTAACTTTTAACAATGTTAAAGATTCAAAAATCTCTGCTGACGGTCACAATG4080
TGACACTAAATAGCAAAGTGAAAACATCTAGCAGCAATGGCGGACGTGAAAGCAATAGCG4140
ACAACGATACCGGCTTAACTATTACTGCAAAAAATGTAGAAGTAAACAAAGATATTACTT4200
CTCTCAAAACAGTAAATATCACCGCGTCGGAAAAGGTTACCACCACAGCAGGCTCGACCA4260
TTAACGCAACAAATGGCAAAGCAAGTATTACAACCAAAACAGGTGATATCAGCGGTACGA4320
TTTCCGGTAACACGGTAAGTGTTAGCGCGACTGGTGATTTAACCACTAAATCCGGCTCAA4380
AAATTGAAGCGAAATCGGGTGAGGCTAATGTAACAAGTGCAACAGGTACAATTGGCGGTA4440
CAATTTCCGGTAATACGGTAAATGTTACGGCAAACGCTGGCGATTTAACAGTTGGGAATG4500
GCGCAGAAATTAATGCGACAGAAGGAGCTGCAACCTTAACCGCAACAGGGAATACCTTGA4560
CTACTGAAGCCGGTTCTAGCATCACTTCAACTAAGGGTCAGGTAGACCTCTTGGCTCAGA4620
ATGGTAGCATCGCAGGAAGCATTAATGCTGCTAATGTGACATTAAATACTACAGGCACCT4680
TAACCACCGTGGCAGGCTCGGATATTAAAGCAACCAGCGGCACCTTGGTTATTAACGCAA4740
AAGATGCTAAGCTAAATGGTGATGCATCAGGTGATAGTACAGAAGTGAATGCAGTCAACG4800
ACTGGGGATTTGGTAGTGTGACTGCGGCAACCTCAAGCAGTGTGAATATCACTGGGGATT4860
TAAACACAGTAAATGGGTTAAATATCATTTCGAAAGATGGTAGAAACACTGTGCGCTTAA4920
GAGGCAAGGAAATTGAGGTGAAATATATCCAGCCAGGTGTAGCAAGTGTAGAAGAAGTAA4980
TTGAAGCGAAACGCGTCCTTGAAAAAGTAAAAGATTTATCTGATGAAGAAAGAGAAACAT5040
TAGCTAAACTTGGTGTAAGTGCTGTACGTTTTGTTGAGCCAAATAATACAATTACAGTCA5100
ATACACAAAATGAATTTACAACCAGACCGTCAAGTCAAGTGATAATTTCTGAAGGTAAGG5160
CGTGTTTCTCAAGTGGTAATGGCGCACGAGTATGTACCAATGTTGCTGACGATGGACAGC5220
CGTAGTCAGTAATTGACAAGGTAGATTTCATCCTGCAATGAAGTCATTTTATTTTCGTAT5280
TATTTACTGTGTGGGTTAAAGTTCAGTACGGGCTTTACCCATCTTGTAAAAAATTACGGA5340
GAATACAATAAAGTATTTTTAACAGGTTATTATTATGAAAAATATAAAAAGCAGATTAAA5400
ACTCAGTGCAATATCAGTATTGCTTGGCCTGGCTTCTTCATCATTGTATGCAGAAGAAGC5460
GTTTTTAGTAAAAGGCTTTCAGTTATCTGGTGCACTTGAAACTTTAAGTGAAGACGCCCA5520
ACTGTCTGTAGCAAAATCTTTATCTAAATACCAAGGCTCGCAAACTTTAACAAACCTAAA5580
AACAGCACAGCTTGAATTACAGGCTGTGCTAGATAAGATTGAGCCAAATAAATTTGATGT5640
GATATTGCCGCAACAAACCATTACGGATGGCAATATCATGTTTGAGCTAGTCTCGAAATC5700
AGCCGCAGAAAGCCAAGTTTTTTATAAGGCGAGCCAGGGTTATAGTGAAGAAAATATCGC5760
TCGTAGCCTGCCATCTTTGAAACAAGGAAAAGTGTATGAAGATGGTCGTCAGTGGTTCGA5820
TTTGCGTGAATTTAATATGGCAAAAGAAAACCCGCTTAAGGTTACCCGTGTACATTACGA5880
ACTAAACCCTAAAAACAAAACCTCTAATTTGATAATTGCGGGCTTCTCGCCTTTTGGTAA5940
AACGCGTAGCTTTATTTCTTATGATAATTTCGGCGCGAGAGAGTTTAACTACCAACGTGT6000
AAGCTTGGGTTTTGTTAATGCCAATTTAACTGGTCATGATGATGTGTTAATTATACCAGT6060
ATGAGTTATGCTGATTCTAATGATATCGACGGCTTACCAAGTGCGATTAATCGTAAATTA6120
TCAAAAGGTCAATCTATCTCTGCGAATCTGAAATGGAGTTATTATCTCCCAACATTTAAC6180
CTTGGCATGGAAGACCAATTTAAAATTAATTTAGGCTACAACTACCGCCATATTAATCAA6240
ACCTCCGCGTTAAATCGCTTGGGTGAAACGAAGAAAAAATTTGCAGTATCAGGCGTAAGT6300
GCAGGCATTGATGGACATATCCAATTTACCCCTAAAACAATCTTTAATATTGATTTAACT6360
CATCATTATTACGCGAGTAAATTACCAGGCTCTTTTGGAATGGAGCGCATTGGCGAAACA6420
TTTAATCGCAGCTATCACATTAGCACAGCCAGTTTAGGGTTGAGTCAAGAGTTTGCTCAA6480
GGTTGGCATTTTAGCAGTCAATTATCAGGTCAATTTACTCTACAAGATATTAGCAGTATA6540
GATTTATTCTCTGTAACAGGTACTTATGGCGTCAGAGGCTTTAAATACGGCGGTGCAAGT6600
GGTGAGCGCGGTCTTGTATGGCGTAATGAATTAAGTATGCCAAAATACACCCGCTTCCAA6660
ATCAGCCCTTATGCGTTTTATGATGCAGGTCAGTTCCGTTATAATAGCGAAAATGCTAAA6720
ACTTACGGCGAAGATATGCACACGGTATCCTCTGCGGGTTTAGGCATTAAAACCTCTCCT6780
ACACAAAACTTAAGCCTAGATGCTTTTGTTGCTCGTCGCTTTGCAAATGCCAATAGTGAC6840
AATTTGAATGGCAACAAAAAACGCACAAGCTCACCTACAACCTTCTGGGGGAGATTAACA6900
TTCAGTTTCTAACCCTGAAATTTAATCAACTGGTAAGCGTTCCGCCTACCAGTTTATAAC6960
TATATGCTTTACCCGCCAATTTACAGTCTATAGGCAACCCTGTTTTTACCCTTATATATC7020
AAATAAACAAGCTAAGCTGAGCTAAGCAAACCAAGCAAACTCAAGCAAGCCAAGTAATAC7080
TAAAAAAACAATTTATATGATAAACTAAAGTATACTCCATGCCATGGCGATACAAGGGAT7140
TTAATAATATGACAAAAGAAAATTTGCAAAACGCTCCTCAAGATGCGACCGCTTTACTTG7200
CGGAATTAAGCAACAATCAAACTCCCCTGCGAATATTTAAACAACCACGCAAGCCCAGCC7260
TATTACGCTTGGAACAACATATCGCAAAAAAAGATTATGAGTTTGCTTGTCGTGAATTAA7320
TGGTGATTCTGGAAAAAATGGACGCTAATTTTGGAGGCGTTCACGATATTGAATTTGACG7380
CACCCGCTCAGCTGGCATATCTACCCGAAAAATTACTAATTTATTTTGCCACTCGTCTCG7440
CTAATGCAATTACAACACTCTTTTCCGACCCCGAATTGGCAATTTCTGAAGAAGGGGCGT7500
TAAAGATGATTAGCCTGCAACGCTGGTTGACGCTGATTTTTGCCTCTTCCCCCTACGTTA7560
ACGCAGACCATATTCTCAATAAATATAATATCAACCCAGATTCCGAAGGTGGCTTTCATT7620
TAGCAACAGACAACTCTTCTATTGCTAAATTCTGTATTTTTTACTTACCCGAATCCAATG7680
TCAATATGAGTTTAGATGCGTTATGGGCAGGGAATCAACAACTTTGTGCTTCATTGTGTT7740
TTGCGTTGCAGTCTTCACGTTTTATTGGTACCGCATCTGCGTTTCATAAAAGAGCGGTGG7800
TTTTACAGTGGTTTCCTAAAAAACTCGCCGAAATTGCTAATTTAGATGAATTGCCTGCAA7860
ATATCCTTCATGATGTATATATGCACTGCAGTTATGATTTAGCAAAAAACAAGCACGATG7920
TTAAGCGTCCATTAAACGAACTTGTCCGCAAGCATATCCTCACGCAAGGATGGCAAGACC7980
GCTACCTTTACACCTTAGGTAAAAAGGACGGCAAACCTGTGATGATGGTACTGCTTGAAC8040
ATTTTAATTCGGGACATTCGATTTATCGTACACATTCAACTTCAATGATTGCTGCTCGAG8100
AAAAATTCTATTTAGTCGGCTTAGGCCATGAGGGCGTTGATAAAATAGGTCGAGAAGTGT8160
TTGACGAGTTCTTTGAAATCAGTAGCAATAATATAATGGAGAGACTGTTTTTTATCCGTA8220
AACAGTGCGAAACTTTCCAACCCGCAGTGTTCTATATGCCAAGCATTGGCATGGATATTA8280
CCACGATTTTTGTGAGCAACACTCGGCTTGCCCCTATTCAAGCTGTAGCCCTGGGTCATC8340
CTGCCACTACGCATTCTGAATTTATTGATTATGTCATCGTAGAAGATGATTATGTGGGCA8400
GTGAAGATTGTTTCAGCGAAACCCTTTTACGCTTACCCAAAGATGCCCTACCTTATGTAC8460
CTTCTGCACTCGCCCCACAAAAAGTGGATTATGTACTCAGGGAAAACCCTGAAGTAGTCA8520
ATATCGGTATTGCCGCTACCACAATGAAATTAAACCCTGAATTTTTGCTAACATTGCAAG8580
AAATCAGAGATAAAGCTAAAGTCAAAATACATTTTCATTTCGCACTTGGACAATCAACAG8640
GCTTGACACACCCTTATGTCAAATGGTTTATCGAAAGCTATTTAGGTGACGATGCCACTG8700
CACATCCCCACGCACCTTATCACGATTATCTGGCAATATTGCGTGATTGCGATATGCTAC8760
TAAATCCGTTTCCTTTCGGTAATACTAACGGCATAATTGATATGGTTACATTAGGTTTAG8820
TTGGTGTATGCAAAACGGGGGATGAAGTACATGAACATATTGATGAAGGTCTGTTTAAAC8880
GCTTAGGACTACCAGAATGGCTGATAGCCGACACACGAGAAACATATATTGAATGTGCTT8940
TGCGTCTAGCAGAAAACCATCAAGAACGCCTTGAACTCCGTCGTTACATCATAGAAAACA9000
ACGGCTTACAAAAGCTTTTTACAGGCGACCCTCGTCCATTGGGCAAAATACTGCTTAAGA9060
AAACAAATGAATGGAAGCGGAAGCACTTGAGTAAAAAATAACGGTTTTTTAAAGTAAAAG9120
TGCGGTTAATTTTCAAAGCGTTTTAAAAACCTCTCAAAAATCAACCGCACTTTTATCTTT9180
ATAACGATCCCGCACGCTGACAGTTTATCAGCCTCCCGCCATAAAACTCCGCCTTTCATG9240
GCGGAGATTTTAGCCAAAACTGGCAGAAATTAAAGGCTAAAATCACCAAATTGCACCACA9300
AAATCACCAATACCCACAAAAAA9323
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4287 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GATCAATCTGGGCGATATTTTTGCCAAAGGTGGTAACATTAATGTCCGCGCTGCCACTAT60
TCGCAATAAAGGTAAACTTTCTGCCGACTCTGTAAGCAAAGATAAAAGTGGTAACATTGT120
TCTCTCTGCCAAAGAAGGTGAAGCGGAAATTGGCGGTGTAATTTCCGCTCAAAATCAGCA180
AGCCAAAGGTGGTAAGTTGATGATTACAGGCGATAAAGTTACATTGAAAACGGGTGCACT240
TATCGACCTTTCGGGTAAAGAAGGGGGAGAAACTTATCTTGGCGGTGACGAGCGTGGCGA300
AGGTAAAAACGGCATTCAATTAGCAAAGAAAACCACTTTAGAAAAAGGCTCAACAATTAA360
TGTGTCAGGTAAAGAAAAAGCTGGGCGCGCTATTGTATGGGGCGATATTGCGTTAATTGA420
CGGCAATATTAATGCCCAAGGTAAAGATATCGCTAAAACTGGTGGTTTTGTGGAGACGTC480
GGGGCATTACTTATCCATTGATGATAACGCAATTGTTAAAACAAAAGAATGGCTACTAGA540
CCCAGAGAATGTGACTATTGAAGCTCCTTCCGCTTCTCGCGTCGAGCTGGGTGCCGATAG600
GAATTCCCACTCGGCAGAGGTGATAAAAGTGACCCTAAAAAAAAATAACACCTCCTTGAC660
AACACTAACCAATACAACCATTTCAAATCTTCTGAAAAGTGCCCACGTGGTGAACATAAC720
GGCAAGGAGAAAACTTACCGTTAATAGCTCTATCAGTATAGAAAGAGGCTCCCACTTAAT780
TCTCCACAGTGAAGGTCAGGGCGGTCAAGGTGTTCAGATTGATAAAGATATTACTTCTGA840
AGGCGGAAATTTAACCATTTATTCTGGCGGATGGGTTGATGTTCATAAAAATATTACGCT900
TGGTAGCGGCTTTTTAAACATCACAACTAAAGAAGGAGATATCGCCTTCGAAGACAAGTC960
TGGACGGAACAACCTAACCATTACAGCCCAAGGGACCATCACCTCAGGTAATAGTAACGG1020
CTTTAGATTTAACAACGTCTCTCTAAACAGCCTTGGCGGAAAGCTGAGCTTTACTGACAG1080
CAGAGAGGACAGAGGTAGAAGAACTAAGGGTAATATCTCAAACAAATTTGACGGAACGTT1140
AAACATTTCCGGAACTGTAGATATCTCAATGAAAGCACCCAAAGTCAGCTGGTTTTACAG1200
AGACAAAGGACGCACCTACTGGAACGTAACCACTTTAAATGTTACCTCGGGTAGTAAATT1260
TAACCTCTCCATTGACAGCACAGGAAGTGGCTCAACAGGTCCAAGCATACGCAATGCAGA1320
ATTAAATGGCATAACATTTAATAAAGCCACTTTTAATATCGCACAAGGCTCAACAGCTAA1380
CTTTAGCATCAAGGCATCAATAATGCCCTTTAAGAGTAACGCTAACTACGCATTATTTAA1440
TGAAGATATTTCAGTCTCAGGGGGGGGTAGCGTTAATTTCAAACTTAACGCCTCATCTAG1500
CAACATACAAACCCCTGGCGTAATTATAAAATCTCAAAACTTTAATGTCTCAGGAGGGTC1560
AACTTTAAATCTCAAGGCTGAAGGTTCAACAGAAACCGCTTTTTCAATAGAAAATGATTT1620
AAACTTAAACGCCACCGGTGGCAATATAACAATCAGACAAGTCGAGGGTACCGATTCACG1680
CGTCAACAAAGGTGTCGCAGCCAAAAAAAACATAACTTTTAAAGGGGGTAATATCACCTT1740
CGGCTCTCAAAAAGCCACAACAGAAATCAAAGGCAATGTTACCATCAATAAAAACACTAA1800
CGCTACTCTTCGTGGTGCGAATTTTGCCGAAAACAAATCGCCTTTAAATATAGCAGGAAA1860
TGTTATTAATAATGGCAACCTTACCACTGCCGGCTCCATTATCAATATAGCCGGAAATCT1920
TACTGTTTCAAAAGGCGCTAACCTTCAAGCTATAACAAATTACACTTTTAATGTAGCCGG1980
CTCATTTGACAACAATGGCGCTTCAAACATTTCCATTGCCAGAGGAGGGGCTAAATTTAA2040
AGATATCAATAACACCAGTAGCTTAAATATTACCACCAACTCTGATACCACTTACCGCAC2100
CATTATAAAAGGCAATATATCCAACAAATCAGGTGATTTGAATATTATTGATAAAAAAAG2160
CGACGCTGAAATCCAAATTGGCGGCAATATCTCACAAAAAGAAGGCAATCTCACAATTTC2220
TTCTGATAAAGTAAATATTACCAATCAGATAACAATCAAAGCAGGCGTTGAAGGGGGGCG2280
TTCTGATTCAAGTGAGGCAGAAAATGCTAACCTAACTATTCAAACCAAAGAGTTAAAATT2340
GGCAGGAGACCTAAATATTTCAGGCTTTAATAAAGCAGAAATTACAGCTAAAAATGGCAG2400
TGATTTAACTATTGGCAATGCTAGCGGTGGTAATGCTGATGCTAAAAAAGTGACTTTTGA2460
CAAGGTTAAAGATTCAAAAATCTCGACTGACGGTCACAATGTAACACTAAATAGCGAAGT2520
GAAAACGTCTAATGGTAGTAGCAATGCTGGTAATGATAACAGCACCGGTTTAACCATTTC2580
CGCAAAAGATGTAACGGTAAACAATAACGTTACCTCCCACAAGACAATAAATATCTCTGC2640
CGCAGCAGGAAATGTAACAACCAAAGAAGGCACAACTATCAATGCAACCACAGGCAGCGT2700
GGAAGTAACTGCTCAAAATGGTACAATTAAAGGCAACATTACCTCGCAAAATGTAACAGT2760
GACAGCAACAGAAAATCTTGTTACCACAGAGAATGCTGTCATTAATGCAACCAGCGGCAC2820
AGTAAACATTAGTACAAAAACAGGGGATATTAAAGGTGGAATTGAATCAACTTCCGGTAA2880
TGTAAATATTACAGCGAGCGGCAATACACTTAAGGTAAGTAATATCACTGGTCAAGATGT2940
AACAGTAACAGCGGATGCAGGAGCCTTGACAACTACAGCAGGCTCAACCATTAGTGCGAC3000
AACAGGCAATGCAAATATTACAACCAAAACAGGTGATATCAACGGTAAAGTTGAATCCAG3060
CTCCGGCTCTGTAACACTTGTTGCAACTGGAGCAACTCTTGCTGTAGGTAATATTTCAGG3120
TAACACTGTTACTATTACTGCGGATAGCGGTAAATTAACCTCCACAGTAGGTTCTACAAT3180
TAATGGGACTAATAGTGTAACCACCTCAAGCCAATCAGGCGATATTGAAGGTACAATTTC3240
TGGTAATACAGTAAATGTTACAGCAAGCACTGGTGATTTAACTATTGGAAATAGTGCAAA3300
AGTTGAAGCGAAAAATGGAGCTGCAACCTTAACTGCTGAATCAGGCAAATTAACCACCCA3360
AACAGGCTCTAGCATTACCTCAAGCAATGGTCAGACAACTCTTACAGCCAAGGATAGCAG3420
TATCGCAGGAAACATTAATGCTGCTAATGTGACGTTAAATACCACAGGCACTTTAACTAC3480
TACAGGGGATTCAAAGATTAACGCAACCAGTGGTACCTTAACAATCAATGCAAAAGATGC3540
CAAATTAGATGGTGCTGCATCAGGTGACCGCACAGTAGTAAATGCAACTAACGCAAGTGG3600
CTCTGGTAACGTGACTGCGAAAACCTCAAGCAGCGTGAATATCACCGGGGATTTAAACAC3660
AATAAATGGGTTAAATATCATTTCGGAAAATGGTAGAAACACTGTGCGCTTAAGAGGCAA3720
GGAAATTGATGTGAAATATATCCAACCAGGTGTAGCAAGCGTAGAAGAGGTAATTGAAGC3780
GAAACGCGTCCTTGAGAAGGTAAAAGATTTATCTGATGAAGAAAGAGAAACACTAGCCAA3840
ACTTGGTGTAAGTGCTGTACGTTTCGTTGAGCCAAATAATGCCATTACGGTTAATACACA3900
AAACGAGTTTACAACCAAACCATCAAGTCAAGTGACAATTTCTGAAGGTAAGGCGTGTTT3960
CTCAAGTGGTAATGGCGCACGAGTATGTACCAATGTTGCTGACGATGGACAGCAGTAGTC4020
AGTAATTGACAAGGTAGATTTCATCCTGCAATGAAGTCATTTTATTTTCGTATTATTTAC4080
TGTGTGGGTTAAAGTTCAGTACGGGCTTTACCCACCTTGTAAAAAATTACGAAAAATACA4140
ATAAAGTATTTTTAACAGGTTATTATTATGAAAAACATAAAAAGCAGATTAAAACTCAGT4200
GCAATATCAATATTGCTTGGCTTGGCTTCTTCATCGACGTATGCAGAAGAAGCGTTTTTA4260
GTAAAAGGCTTTCAGTTATCTGGCGCG4287
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4702 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGGAATGAGCGTCGTACACGGTACAGCAACCATGCAAGTAGACGGCAATAAAACCACTAT60
CCGTAATAGCATCAATGCTATCATCAATTGGAAACAATTTAACATTGACCAAAATGAAAT120
GGAGCAGTTTTTACAAGAAAGCAGCAACTCTGCCGTTTTCAACCGTGTTACATCTGACCA180
AATCTCCCAATTAAAAGGGATTTTAGATTCTAACGGACAAGTCTTTTTAATCAACCCAAA240
TGGTATCACAATAGGTAAAGACGCAATTATTAACACTAATGGCTTTACTGCTTCTACGCT300
AGACATTTCTAACGAAAACATCAAGGCGCGTAATTTCACCCTTGAGCAAACCAAGGATAA360
AGCACTCGCTGAAATCGTGAATCACGGTTTAATTACCGTTGGTAAAGACGGTAGCGTAAA420
CCTTATTGGTGGCAAAGTGAAAAACGAGGGCGTGATTAGCGTAAATGGCGGTAGTATTTC480
TTTACTTGCAGGGCAAAAAATCACCATCAGCGATATAATAAATCCAACCATCACTTACAG540
CATTGCTGCACCTGAAAACGAAGCGATCAATCTGGGCGATATTTTTGCCAAAGGTGGTAA600
CATTAATGTCCGCGCTGCCACTATTCGCAATAAAGGTAAACTTTCTGCCGACTCTGTAAG660
CAAAGATAAAAGTGGTAACATTGTTCTCTCTGCCAAAGAAGGTGAAGCGGAAATTGGCGG720
TGTAATTTCCGCTCAAAATCAGCAAGCCAAAGGTGGTAAGTTGATGATTACAGGTGATAA780
AGTCACATTAAAAACAGGTGCAGTTATCGACCTTTCAGGTAAAGAAGGGGGAGAGACTTA840
TCTTGGCGGTGATGAGCGTGGCGAAGGTAAAAATGGTATTCAATTAGCGAAGAAAACCTC900
TTTAGAAAAAGGCTCGACAATTAATGTATCAGGCAAAGAAAAAGGCGGGCGCGCTATTGT960
ATGGGGCGATATTGCATTAATTAATGGTAACATTAATGCTCAAGGTAGCGATATTGCTAA1020
AACTGGCGGCTTTGTGGAAACATCAGGACATGACTTATCCATTGGTGATGATGTGATTGT1080
TGACGCTAAAGAGTGGTTATTAGACCCAGATGATGTGTCCATTGAAACTCTTACATCTGG1140
ACGCAATAATACCGGCGAAAACCAAGGATATACAACAGGAGATGGGACTAAAGAGTCACC1200
TAAAGGTAATAGTATTTCTAAACCTACATTAACAAACTCAACTCTTGAGCAAATCCTAAG1260
AAGAGGTTCTTATGTTAATATCACTGCTAATAATAGAATTTATGTTAATAGCTCCATCAA1320
CTTATCTAATGGCAGTTTAACACTTCACACTAAACGAGATGGAGTTAAAATTAACGGTGA1380
TATTACCTCAAACGAAAATGGTAATTTAACCATTAAAGCAGGCTCTTGGGTTGATGTTCA1440
TAAAAACATCACGCTTGGTACGGGTTTTTTCAATATTGTCGCTGGGGATTCTGTAGCTTT1500
TGAGAGAGAGGGCGATAAAGCACGTAACGCAACAGATGCTCAAATTACCGCACAAGGGAC1560
GATAACCGTCAATAAAGATGATAAACAATTTAGATTCAATAATGTATCTATTAACGGGAC1620
GGGCAAGGGTTTAAAGTTTATTGCAAATCAAAATAATTTCACTCATAAATTTGATGGCGA1680
AATTAACATATCTGGAATAGTAACAATTAACCAAACCACGAAAAAAGATGTTAAATACTG1740
GAATGCATCAAAAGACTCTTACTGGAATGTTTCTTCTCTTACTTTGAATACGGTGCAAAA1800
ATTTACCTTTATAAAATTCGTTGATAGCGGCTCAAATTCCCAAGATTTGAGGTCATCACG1860
TAGAAGTTTTGCAGGCGTACATTTTAACGGCATCGGAGGCAAAACAAACTTCAACATCGG1920
AGCTAACGCAAAAGCCTTATTTAAATTAAAACCAAACGCCGCTACAGACCCAAAAAAAGA1980
ATTACCTATTACTTTTAACGCCAACATTACAGCTACCGGTAACAGTGATAGCTCTGTGAT2040
GTTTGACATACACGCCAATCTTACCTCTAGAGCTGCCGGCATAAACATGGATTCAATTAA2100
CATTACCGGCGGGCTTGACTTTTCCATAACATCCCATAATCGCAATAGTAATGCTTTTGA2160
AATCAAAAAAGACTTAACTATAAATGCAACTGGCTCGAATTTTAGTCTTAAGCAAACGAA2220
AGATTCTTTTTATAATGAATACAGCAAACACGCCATTAACTCAAGTCATAATCTAACCAT2280
TCTTGGCGGCAATGTCACTCTAGGTGGGGAAAATTCAAGCAGTAGCATTACGGGCAATAT2340
CAATATCACCAATAAAGCAAATGTTACATTACAAGCTGACACCAGCAACAGCAACACAGG2400
CTTGAAGAAAAGAACTCTAACTCTTGGCAATATATCTGTTGAGGGGAATTTAAGCCTAAC2460
TGGTGCAAATGCAAACATTGTCGGCAATCTTTCTATTGCAGAAGATTCCACATTTAAAGG2520
AGAAGCCAGTGACAACCTAAACATCACCGGCACCTTTACCAACAACGGTACCGCCAACAT2580
TAATATAAAACAAGGAGTGGTAAAACTCCAAGGCGATATTATCAATAAAGGTGGTTTAAA2640
TATCACTACTAACGCCTCAGGCACTCAAAAAACCATTATTAACGGAAATATAACTAACGA2700
AAAAGGCGACTTAAACATCAAGAATATTAAAGCCGACGCCGAAATCCAAATTGGCGGCAA2760
TATCTCACAAAAAGAAGGCAATCTCACAATTTCTTCTGATAAAGTAAATATTACCAATCA2820
GATAACAATCAAAGCAGGCGTTGAAGGGGGGCGTTCTGATTCAAGTGAGGCAGAAAATGC2880
TAACCTAACTATTCAAACCAAAGAGTTAAAATTGGCAGGAGACCTAAATATTTCAGGCTT2940
TAATAAAGCAGAAATTACAGCTAAAAATGGCAGTGATTTAACTATTGGCAATGCTAGCGG3000
TGGTAATGCTGATGCTAAAAAAGTGACTTTTGACAAGGTTAAAGATTCAAAAATCTCGAC3060
TGACGGTCACAATGTAACACTAAATAGCGAAGTGAAAACGTCTAATGGTAGTAGCAATGC3120
TGGTAATGATAACAGCACCGGTTTAACCATTTCCGCAAAAGATGTAACGGTAAACAATAA3180
CGTTACCTCCCACAAGACAATAAATATCTCTGCCGCAGCAGGAAATGTAACAACCAAAGA3240
AGGCACAACTATCAATGCAACCACAGGCAGCGTGGAAGTAACTGCTCAAAATGGTACAAT3300
TAAAGGCAACATTACCTCGCAAAATGTAACAGTGACAGCAACAGAAAATCTTGTTACCAC3360
AGAGAATGCTGTCATTAATGCAACCAGCGGCACAGTAAACATTAGTACAAAAACAGGGGA3420
TATTAAAGGTGGAATTGAATCAACTTCCGGTAATGTAAATATTACAGCGAGCGGCAATAC3480
ACTTAAGGTAAGTAATATCACTGGTCAAGATGTAACAGTAACAGCGGATGCAGGAGCCTT3540
GACAACTACAGCAGGCTCAACCATTAGTGCGACAACAGGCAATGCAAATATTACAACCAA3600
AACAGGTGATATCAACGGTAAAGTTGAATCCAGCTCCGGCTCTGTAACACTTGTTGCAAC3660
TGGAGCAACTCTTGCTGTAGGTAATATTTCAGGTAACACTGTTACTATTACTGCGGATAG3720
CGGTAAATTAACCTCCACAGTAGGTTCTACAATTAATGGGACTAATAGTGTAACCACCTC3780
AAGCCAATCAGGCGATATTGAAGGTACAATTTCTGGTAATACAGTAAATGTTACAGCAAG3840
CACTGGTGATTTAACTATTGGAAATAGTGCAAAAGTTGAAGCGAAAAATGGAGCTGCAAC3900
CTTAACTGCTGAATCAGGCAAATTAACCACCCAAACAGGCTCTAGCATTACCTCAAGCAA3960
TGGTCAGACAACTCTTACAGCCAAGGATAGCAGTATCGCAGGAAACATTAATGCTGCTAA4020
TGTGACGTTAAATACCACAGGCACTTTAACTACTACAGGGGATTCAAAGATTAACGCAAC4080
CAGTGGTACCTTAACAATCAATGCAAAAGATGCCAAATTAGATGGTGCTGCATCAGGTGA4140
CCGCACAGTAGTAAATGCAACTAACGCAAGTGGCTCTGGTAACGTGACTGCGAAAACCTC4200
AAGCAGCGTGAATATCACCGGGGATTTAAACACAATAAATGGGTTAAATATCATTTCGGA4260
AAATGGTAGAAACACTGTGCGCTTAAGAGGCAAGGAAATTGATGTGAAATATATCCAACC4320
AGGTGTAGCAAGCGTAGAAGAGGTAATTGAAGCGAAACGCGTCCTTGAGAAGGTAAAAGA4380
TTTATCTGATGAAGAAAGAGAAACACTAGCCAAACTTGGTGTAAGTGCTGTACGTTTCGT4440
TGAGCCAAATAATGCCATTACGGTTAATACACAAAACGAGTTTACAACCAAACCATCAAG4500
TCAAGTGACAATTTCTGAAGGTAAGGCGTGTTTCTCAAGTGGTAATGGCGCACGAGTATG4560
TACCAATGTTGCTGACGATGGACAGCAGTAGTCAGTAATTGACAAGGTAGATTTCATCCT4620
GCAATGAAGTCATTTTATTTTCGTATTATTTACTGTGTGGGTTAAAGTTCAGTACGGGCT4680
TTACCCACCTTGTAAAAAATTA4702
__________________________________________________________________________
Claims
1. A vaccine against disease caused by non-typeable Haemophilus influenzae, including otitis media, sinusitis and bronchitis, comprising an effective amount of a high molecular weight protein of non-typeable Haemophilus influenzae which is protein HMW1 and/or HMW2 and a physiological carrier therefor.
2. The vaccine of claim 1 wherein said protein is HMW1 encoded by the DNA sequence shown in FIG. 1 (SEQ ID NO:1), having the derived amino acid sequence of FIG. 2 (SEQ ID ID NO:2) and having an apparent molecular weight of 125 kDa.
3. The vaccine of claim 1 wherein said protein is HMW2 encoding by the DNA sequence shown in FIG. 3 SEQ ID NO:3), having the derived amino acid sequence of FIG. 4 SEQ ID NO:4) and having an apparent molecular weight of 120 kDa.
- Barenkamp S. J., Pediatr Res 29(4 part 2) 1991 p. 167A Abstract #985. Van Regenmortel, Immunology Today 10(8):266-272, 1989. Dick et al, Contrib Microbiol. Immunol 10:48-114, 1989. Roitt et al, eds Immunology, C. V. Mosby Co, St. Louis, Gowe-Medical Publishing, London, 1985 pp. 8.3-8.4. Boslego et al Vaccine 9:154-162, 1991. Pediatr. Infect. Dis. J., 9:333-339, 1990. The Journal of Infectious Diseases, vol. 165(Suppl.), issued Aug. 1992, S. J. Barenkamp, "Outer Membrane Protein and Lipopolysaccharides of Nontypeable Haemophilus influenzae", pp. S181-S184, see entire document. Infection and Immunity, vol. 56(1), issued Jan. 1988, E. J. Hansen, "Immune Enhancement of Pulmonary Clearance on Nontypable Haemophilus influenzae,"pp. 182-190, see entire document, especially Figs. 3 and 4. Infection and Immunity, vol. 60(4), issued Apr. 1992, S. J. Barenkamp et al, "Cloning, Expression and DNA Sequence Analysis of Genes Encoding Nontypeable Haemophilus influenzae High -Molecular-Weight Surface-Exposed Proteins Related to Filamentous Hemagglutinin of Bordetella pertussis," pp. 1302-1313, see entire document. Infection and Immunity, vol. 52(2), issued May 1986, S. J. Barenkamp, "Protection by Serum Antibodies in Experimental Nontypable Haemphilus influenzae Otitis Media", pp. 572-578, see Figs. 1 and 2. Proceedings of the National Academy of Sciences USA, vol. 80, issued Mar. 1983, R. A. Young et al, "Efficient Isolation of Genes by Using Antibody Probes, "pp. 1194-1198, see entire document. Journal of Molecular Biology, vol. 157, issued 1982, J. Kyte et al, "A Simple Method for Displaying the Hydrophatic Character of a Protein", pp. 105-132, see entire document. Proceedings of the National Academy of Sciences, vol. 78(6), issued Jun. 1981, T. P. Hopp et al, "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", pp. 3824-3828, see entire document. Infection and Immunity, vol. 45(3), issued Sep. 1984, R. Schneerson et al, "Serum Antibody Responses of Juvenile and Infant Rhesus Monkeys Injected with Haemophilus influenzae Type b and Pneumoccus Type 6A Capsular Polysaccharide-Protein Conjugates", pp. 582-591, see entire document.
Type: Grant
Filed: Mar 16, 1993
Date of Patent: Aug 27, 1996
Assignees: St. Louis University (St. Louis, MO), Washington University (St. Louis, MO)
Inventors: Stephen J. Barenkamp (Webster Grove, MO), Joseph W. St. Geme, III (St. Louis, MO)
Primary Examiner: James C. Housel
Assistant Examiner: Julie Krsek-Staples
Law Firm: Shoemaker and Mattare, Ltd.
Application Number: 8/38,682
International Classification: A61K 39102; A61K 3816;
